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Transcriptional and Posttranscriptional Mechanisms Modulate Creatine Kinase Expression during Differentiation of Osteoblastic Cells*

Enhancement of energy metabolism is fundamental to the developmental programs of many cell types. This work examines the molecular mechanisms that mediate changes in energy metabolism during differentiation of osteoblastic cells. When the rat osteoblastic cell line, ROS 17/2.8, is induced to differentiate with 1,25-dihy-droxyvitamin D,, expression of creatine kinase-b (ck-b), a pivotal enzyme in energy metabolism, is enhanced. Maximum enhancement occurs at 48 h of induction with 10 n~ 1,26-dihydroxyvitamin D, when creatine kinase activity is 2.1-fold over uninduced cells. This is associ- ated with a 2-fold increase in transcription rate and the formation of a second protein-DNA complex on the ck-b gene promoter that is supplementary to the one present in undifferentiated cells. In addition, the contribution of posttranscriptional regulatory mechanisms is suggested by (1) the increase in ck-b mRNA abundance exceeds that of transcription rate, indicating an increase in message stability, (2) the increase in ck-b mRNA precedes and exceeds that of protein activity, indicating translational modulation, and (3) RNA mobility-shift assays in- dicate that a cytosolic factor in ROS 17/23 cells interacts specifically with the highly conserved 3”untranslated region of the ck-b mRNA. We have previously reported that such an interaction mediates translational control (Ch’ng, J. L. C., Shoemaker, D. L., Schimmel, P., and Holmes, E. W. (1990) Science 248,1003-1006). The physio-logical

Enhancement of energy metabolism is an integral component of differentiation in many cell systems. During differentiation of osteoblasts, energy metabolism is up-regulated to meet increased energy demands for functions such as changes in cell shape and enhanced activity of membrane pumps. Changes in cell shape facilitate adherence to bone surfaces and serve the purpose of limiting exposure ofbone to osteoclasts (1). Enhanced activity of membrane pumps is related to the formation of extracellular matrix and mineralization. This increase in energy metabolism is represented by an increase in the specific activity of creatine kinase-b (ck-b),l a pivotal enzyme in cal Nutrition Research Center, March of Dimes Birth Defects Founda-* This work was supported in part by the Vanderbilt University Clinition Grant FY92-1198, and National Institutes of Health Grant l R O l DK44490. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  Hence, it acts as a transducer of high energy phosphate bonds from sites of production to sites of utilization. This ensures that there is no depletion of ATP in subcellular components where energy is being rapidly expanded. The importance of creatine kinase in energy metabolism is seen in the stringent regulation of its expression, both developmentally and spatially. Creatine kinase activity has been shown to be closely associated with the cytoskeleton, providing ATP for microtubular movement (5).
Cytoskeletal changes and alteration in cell shape occur in osteoblasts which have been induced to differentiate with 1,25dihydroxyvitamin D3 (1,25-(OH)2D3) (6). Shapiro and colleagues (7) have demonstrated that the level of ck-b activity is directly related to chondrocyte maturation and a decrease in enzyme activity occurs in vitamin D deficiency. Similar findings were reported by Katoh and colleagues (8). Studies in rats indicate that creatine kinase activity is necessary for proper matrix maturation and mineral deposition (9). Inhibition of enzyme activity by administering 0-guanidinopropionic acid results in profound changes including disorganization of chondrocytes in the proliferative and hypertrophic zones, poor vascularization, changes in the electrophoretic mobility of type I1 and type X collagens and inhibition of apatite formation. Hence, enhancement of ck-b activity is necessary for normal development of bone.
The molecular mechanisms that mediate the up-regulation of energy metabolism during osteoblast differentiation are largely unknown. By studying the regulation of ck-b, a pivotal enzyme in cellular energy metabolism in osteoblasts, insight could be derived on these modulatory mechanisms as well as on differentiation of osteoblasts. For instance, during muscle differentiation, expression of creatine kinase-m, the predominant cytosolic isoenzyme of creatine kinase in muscle, is markedly increased (10,ll). This is mediated by the interaction of MyoD, an essential factor in muscle differentiation, with the promoter of the ck-m gene (12). A similar mechanism may occur during osteoblast differentiation involving the interaction of a transcriptional activator with one of the many positive-acting elements in the ck-b gene promoter (13,14). The known positiveacting elements are mainly 3' of -195 nucleotides (nt) from the transcription start site (15,16). It has been shown that two different RNA polymerase I1 initiation complexes can assemble on the rat ck-b promoter (17). There are several elements that show homology to the responsive elements of known transacting factors including those for MyoD (E-box motif), adenovirus E l a protein, Spl, and CTF-l(l3). An AT-rich sequence at -60 binds the TA-rich recognition protein (TARP) which is found in several tissues and is likely to be a positive-acting transcription factor for the ck-b gene (18). However, it is not developmentally regulated, and thus far, no element has been described that is differentiation-specific. Further, there is no recognizable vitamin D-responsive element.
We have earlier reported that translational regulation of ck-b is significant in monocytes (19). This is mediated by the 3'untranslated region of the ck-b mRNA which is highly conserved across species (>70% for chicken:man) (20). This region of the ck-b mRNA interacts with a cytosolic factor to form a protein-RNA complex at the 3' end of the message. Translation is inhibited when the complex forms; conversely, translation is enhanced when affinity of the cytosolic factor for the cis-acting RNA element decreases, resulting in dissociation of the complex.
This study shows that ck-b is specifically up-regulated during differentiation of osteoblastic cells. This is mediated by an increase in transcription rate and is associated with the formation of an additional protein-DNA complex on the ck-b gene promoter. Evidence is also presented on the interaction of a cytosolic factor in osteoblastic cells with the highly conserved 3'-untranslated region of the ck-b mRNA. It is hypothesized that these mechanisms facilitate the enhancement of ck-b expression as well as its targeting to high energy-expanding subcellular structures in the mature osteoblast. This rat cell line was derived from an osteosarcoma and retained many features of osteoblasts (21). Cells were plated on 10-mm Petri dishes and cultured in Ham's F-12 medium (Life Technologies, Inc.) supplemented with 10% charcoal-stripped fetal calf serum. Incubation with 10 nM 1,25-(OH),D3 (Biomol Research Laboratories, Inc., Plymouth Meeting, PA) was started when cells were -20% confluent. Control cells were incubated with a similar concentration of vehicle (ethanol). The concentration of ethanol in treated and control cells did not exceed 0.1%. Cells were harvested for analysis while in log phase growth (<70% confluent).

MATERIALS AND METHODS
ck-b-specific Activity Assay-A sensitive radiochemical assay was used to measure the specific activity of ck-b (22). We have previously demonstrated that creatine kinase activity measured by this assay parallels creatine kinase protein abundance as measured by Western blotting but is more sensitive (23). The assay measures the creatine kinasecatalyzed conversion of I4C-labeled creatine to creatine phosphate. Labeled creatine phosphate is separated from unlabeled creatine by binding to charged DEAE disks. Formation of creatine phosphate is favored by coupling the reaction to the pyruvate kinase-catalyzed conversion of ADP to ATP in the presence of phosphoenolpyruvate. This assay for creatine kinase activity is highly specific and has a sensitivity of ~0 . 5 milliunit/mg of protein when using [I4C]creatine with a specific activity of >15 m C i / m (Amersham Corp.).
To assay for creatine kinase activity, ROS 1712.8 cells were trypsinized and pelleted by centrifugation. They were then washed with phosphate-buffered saline, re-centrifuged, and lyzed in 50 m~ Tris (pH 8.0) and 1 mM dithiothreitol by three cycles of freeze-thawing in an ethanoldry ice mixture. Soluble cytosolic extract was separated from insoluble cellular components by centrifugation and stored a t 20 "C until assay. Ten micrograms of protein extract, as measured by Bio-Rad protein assay, were added to each reaction mixture to assay for creatine kinase activity.
Creatine Kinase-b mRNA Abundance-ROS 1712.8 cells were incubated with 10 n~ 1,25-(OHI2D3 or vehicle in 10-cm Petri dishes. At timed intervals, total RNA was isolated from the cells with guanidinium thiocyanate and purified by centrifugation through a cesium chloride gradient. ck-b mRNA abundance was assayed by Northern blot analysis (24). Fifteen micrograms of total RNA was electrophoresed on a 1% denaturing formaldehyde-agarose gel and transferred overnight onto a Nytran membrane. The RNA was cross-linked to the membrane by a Stratagene W Stratalinker. The membrane was hybridized with a 32P-labeled probe made by random-priming with rat ck-b cDNA as the template (Multiprime DNA Labeling System, Amersham Corp.). Rat ck-b cDNA was a gift from Dr. Pamela Benfield (E. I. Du Pont, Wilmington, DE). Following overnight hybridization at 42 "C, the Northern blot was washed at high stringency (65 "C, 0 . 1 x SSC) before autoradiography. ck-b mRNA gives a signal at 1.3 kb in size. As control, the filter was simultaneously hybridized to a similarly labeled p-actin probe. This gives a signal that corresponds to 2.2 kb and is easily distinguishable from that of ck-b. p-Actin expression is unaffected by 1,25-(OH)zD3 in ROS 1712.8 cells (25). ck-b mRNA abundance, normalized to that of p-actin in the same sample, was determined by the relative intensities of their signals as measured on a laser densitometer (Pharmacia LKB Biotechnology Inc.). The comparative abundance of ck-b mRNA with and without 1,25-(OH),D3 was hence determined.
ck-b Dunscription Rate-ck-b transcription rate in control and 1,25-(OH),D3-treated ROS 1712.8 cells was determined by nuclear run-on analysis (26). Approximately 2 x lo7 cells were removed from Petri dishes by scraping with a Teflon scraper. The cells were pelleted by centrifugation and lyzed in 4 ml of Nonidet-40 lysis buffer. Nuclei were pelleted by centrifugation at 500 x g a t 4 "C, resuspended in 4 ml of Nonidet-40 lysis buffer, vortexed, and centrifuged again under similar conditions. The nuclear pellet was then resuspended in 200 pl of glycerol storage buffer and stored in liquid nitrogen until assayed.
To perform the run-on assay, nuclei were thawed and incubated with 50 mM ATP, CTP, and GTP, and 100 pCi of [32P1UTP (800 Ci/mM, Amersham Corp.) a t 30 "C, with shaking, for 30 min. RNase-free DNase I and proteinase K (Sigma) were added, phenolkhloroform extraction was performed twice, and labeled RNA was precipitated with ethanol. This was then hybridized to a Nytran membrane to which 25 pg each of rat ck-b cDNA and p-actin cDNA (internal standard) had been crosslinked. The hybridization was done at 55 "C in 50% formamide for 72 h. The filter was washed at high stringency with the last wash being 0.1 x SSC at 65 "C, followed by incubation with 10 pg/ml RNase A in 0.3 M NaCl a t 37 "C for 10 min. The membrane was autoradiographed, and the intensity of the ck-b signal (measured by laser densitometry) relative to that of p-actin was compared between control and 1,25-(OH)zD3treated cells.
DNA-Nuclear Factor Interaction-DNA-nuclear factor interactions were studied by DNAmobility-shift assay using gel electrophoresis (17). By restriction enzyme digestion, a 421-bp DNA fragment, that spans from -192 to +228 n t relative to the transcription start site of the ck-b gene, was obtained. This fragment, which includes a sequence 3' to the start site, contains all of the elements reported to be essential for ck-b gene expression (15.16). It was end-labeled with and incubated with nuclear extracts from control and 1,25-(OH)zD3-treated (10 nM for 48 h) cells. Nuclear extracts were obtained using the protocol of Dignam et al. (27). Briefly, cells were lysed using a Dounce homogenizer, nuclei were precipitated by centrifugation, lysed in high salt buffer and refined by dialysis a t 4 "C for 6 h. Each reaction mixture (total volume = 20 pl) contained 5 pg of nuclear extract and 0.2 ng of labeled DNA sequence (-lo5 cpm). To reduce nonspecific interactions, poly(dI-dC).poly(dI-dC) (Boehringer Mannheim) and bovine serum albumin were added. The mixture was incubated at 30 "C for 15 min before being applied to a 4% non-denaturing, low ionic strength polyacrylamide gel and electrophoresed at 200 V in low ionic-strength buffer. The gel was dried and autoradiographed.
RNA Mobility-shift Assay-Specific interactions between cytosolic factors and the ck-b mRNA were analyzed by RNA mobility-shift assays (19). RNA probes corresponding to the entire 3"untranslated region (195 bases) and adjacent 3"coding region of the ck-b mRNA (55 bases) were transcribed from the pBlueScribe in-vitro system (Stratagene Cloning Systems, San Diego, CA) with T7 RNA polymerase. The probes were labeled with [32PlUTP and purified by denaturing polyacrylamide gel electrophoresis. Twenty nanomoles of RNA probe (lo5 cpm) were incubated with 40 pg of SlOO (cytosolic) extract from control and 1,25-(OH),D3-treated cells at 4 "C for 30 min. RNase T1 (1 unit) and heparin (5 mg/ml) were added sequentially a t 10-min intervals. This helps to increase specificity of the RNA-protein interaction. Protein-RNA complexes were resolved by electrophoresis on a 4% non-denaturing, low cross-linked polyacrylamide gel with 0.5 x TBE as buffer. As a control, an RNA probe of similar length but unrelated sequence was used.

ck-b RNA Abundance following 1,25-(0H)&-Two
separate experiments were done to study changes in mRNA abundance following treatment with 1,25-(OH)2D3. This increase was determined by Northern analysis and the quantitation of ck-b mRNA abundance was normalized to that of p-actin. Harrison and colleagues (25) have shown that the transcription rate and message abundance of ck-b in ROS 17/23 cells is unaffected by 1,25-(OH)2D3 treatment. We have confirmed this finding by comparing the intensity of the autoradiographic signal for p-actin a t various time points of 1,25-(OH)zD3 treatment with the intensity of ethidium bromide staining for 28 S and 18 S ribosomal RNA (data not shown). A 1.3-fold increase in ck-b mRNA abundance was seen a t 6 h of incubation with 1,25-(OH)2D3 (Fig. 2). At 24 and 48 h, the increase in mRNA abundance was 4.6-and 5.7-fold, respectively. The maximum increase occurred a t 48 h and is maintained for the duration of the study (96 h). Hence, the increase in ck-b mRNA abundance preceded and exceeded the increase in protein abundance.
Concomitant with the increase in ck-b mRNA abundance is a 2.0-and 2.6-fold increase in 1,25-(OH)2D3 receptor mRNAat 24 and 48 h, respectively (data not shown). This indicates that the positive feedback mediated by 1,25-(OH)zD3 on the expression of its receptor is functional in these cells. However, there is no element in the ck-b gene promoter that resembles the cognate sequence for the vitamin D receptor. This suggests that the effect of 1,25-(OH)2D3 on ck-b expression in ROS 17/23 cells is indirect and is consequent to its induction of differentiation.
Dunscription Rate of ck-&Transcription rate of ck-b in ROS 17/2.8 cells was measured by nuclear run-on experiments. An internal standard, p-actin, was used for both control and treated cells. Transcription of p-actin, as determined by nuclear run-on assay, is unaltered by 1,25-(OH)2D3 in ROS 17\23 cells (25). Transcription of ck-b in cells treated for 48 h with 10 nM 1,25-(OH)2D3 is increased 2-fold (Fig. 3). As ck-b mRNA abundance peaks a t 48 h and remains at this level for up to 96 h, it is likely that the transcription rate at 48 h is the peak level induced by 1,25-(OH)2D3. Hence, there is a significant difference between the increase in transcription rate and the increase in mRNA abundance a t 48 h of incubation with 1,25-(OH)2D3, the increases being 2-and 5.7-fold, respectively. This suggests that, in addition to transcriptional enhancement, a second mechanism, involving increased ck-b message stability, occurs following differentiation of ROS 17/23 cells.
Nuclear Protein-ck-b Promoter Interactions during Differentiation of ROS 1712. 8 Cells-This was studied by DNA mobility-shift assay using a labeled 421-bp DNA fragment that corresponds in sequence from -192 to +228 nt relative to the transcription start site of the ck-b gene promoter. It has previously been shown that this fragment contains all known positive-acting promoter elements (15,16). The data are shown in Fig. 4. Lanes 1 , 2, and 3 contain nuclear extracts from ROS 17/2.8 cells treated with 10 nM 1,25-(OH)zD3 for 48 h. Lanes 4 and 5 contain extracts from control cells. Lane 1 shows that two (an upper and a lower) nuclear factor-DNA complexes are formed with nuclear extracts from l,25-(OH),D3-treated cells. Both complexes are competed out when >100-fold excess of unlabeled DNA corresponding to the entire 421 bp is added in lune 2. Only the lower complex which is related to 1,25-(OH)zD3 treatment is competed out in lune 3 with a >100-fold excess of an unlabeled 228-bp fragment that corresponds in sequence to +1 to +228 nt. Only the upper complex is present when the labelled promoter is incubated with nuclear extracts from control cells in lane 4. The addition of 1,25-(OH),D3 in vitro to nuclear extracts from control cells did not reproduce the lower complex in lane 5 . These interactions are specific as ( a ) poly(d1-dC).poly(dI-dC) and bovine serum albumin are added to hinder nonspecific interactions and ( b ) both complexes are susceptible to in vitro competition with unlabeled DNA that is similar in sequence as the promoter. Competition studies with unlabeled DNA fragments indicate that the cisacting element for the constitutive complex is within -192 to +1 nt while that for the differentiation-induced complex is within +1 to +228 nt.
These studies demonstrate that the enhancement of ck-b transcription during differentiation is associated with the formation of an additional protein-DNA complex (lower band on gel shift assay) on the ck-b gene promoter. The upper complex which is present in both the control and differentiated cells could be associated with constitutive expression. 1,25-(OH)2& itself does not appear to be a component of the lower complex as its addition in vitro to the reaction mixture containing nuclear extracts from control cells did not reproduce the complex (lane 5). We have also ascertained that the 1,25-(OH)zD3 receptor is not involved in either of the two complexes. This was determined by the use of monocloncal antibodies against the 1,25-(OH)2D3 receptor (antibody 9A7, Affinity BioReagents, Neshanic Station, NJ). Addition of this antibody either before or after the formation of the complex did not affect its mobility or abundance.

Posttranscriptional Regulation of ck-b Expression during Dip ferentiation of ROS 1712.8 Cells-Data from
the studies on ck-b transcription rate, mRNA abundance, and protein activity following differentiation indicate a discrepancy in the degree of enhancement among these indices. The increase in mRNA abundance far exceeds that of transcription rate, implicating the effect of increased message stability. In addition, the effect of translational modulation is implicated by the increase in mRNA abundance preceding and exceeding that in protein activity. An increase in mRNA abundance is already detectable at 6 h of incubation while that of protein is significant only a t 24 h. While both peak a t 48 h, the increase in mRNA abundance exceeds 5-fold while the increase in protein abundance is 2.1-  fold. Data from RNA mobility-shift assay indicate that a cytosolic trans-acting factor binds specifically to the 3"untranslated region of the ck-b mRNA (Fig. 5). This occurs in both control and 1,251-(OH)~D~-treated cells. This interaction has previously been shown to be associated with translational modulation of the ck-b message by preventing completion of translation (19). Formation of such complexes in the 3'-untranslated region of other mRNAs is also associated with increased message stability and translational modulatory events (28).

DISCUSSION
These studies show that ck-b expression in osteoblastic cells is specifically enhanced during 1,25-(OH)2D3-modulated differentiation. Enhancement of expression is time-dependent; it is significant only after 24 h of incubation with 10 nM 1,25-(OH)zD3, peaks a t 48 h with a 2.1-fold increase over control, and remains elevated at the peak level for up to 96 h.
The 2-fold increase in ck-b activity reflects the overall increase in the whole cell. There is convincing evidence in the literature that ck-b expression is not uniform throughout the cell but is concentrated a t subcellular structures where energy is actively expanded (5,(29)(30)(31). This includes the cytoskeleton and membrane channels, two structures in ROS 17/23 cells which show enhanced activity following 1,25-(OH)zD3 treatment. Changes in cytoskeleton in the mature osteoblastic cell facilitate adherence to the bone surface while increased activity of membrane pumps is related to the role of the mature osteoblast in the formation of extracellular matrix. Therefore, it is highly probable that the increase in ck-b expression following 1,25-dihydroxyvitamin D3 is focused on these subcellular compartments. As such, the increase locally will be larger than the 2-fold overall increase detected in the cell as a whole.
Increased abundance of ck-b mRNA precedes and exceeds the increase in protein activity. Transcript abundance is significantly increased at 6 h and peaks at 48 h with a 5.7-fold increase over control. This degree of increase is maintained for as long as 1,25-(OH)zD3 incubation continues. The transcription rate of ck-b, however, is increased only 2-fold at 48 h. Hence, increased transcription can only partially account for the higher mRNA abundance; increased mRNA stability is also implicated. In addition, as the increase in protein is less than that in mRNA abundance, translational modulation is also involved.
At the transcription level, basal ck-b expression in ROS 17/ 2.8 cells is associated with a protein-DNA complex that involves a cis-acting element within -192 to +1 nt of the ck-b promoter. Enhancement of transcription by 1,25-(OH)2D3 is coupled with the formation of a second DNA-protein complex that forms on a different cis-acting element within +1 to +228 nt. This latter complex does not involve nuclear-translocated ligand-bound 1,25-(OH)zD3 receptor (VDR) as no recognizable M R response element is present on the ck-b promoter, and the addition of antibodies against VDR did not cause any change in the DNA mobility-shift assay. Consequently, the effect of 1,25-(OH)2D3 on ck-b expression is most likely indirect, being the outcome of its effects on osteoblast differentiation.
1,25-(OH),D, is known to modulate the expression of several genes, one or more of which may then modulate ck-b expression. This interpretation is consistent with the need for an extended interval of 1,25-(OH).$3 treatment before changes in ck-b expression occur. The ck-b gene promoter is known to be complex. There are multiple potential promoter sequences, and at least two different RNA polymerase complexes can form on the promoter (14,17). Several TA-rich protein-binding regulatory regions have been described for the ck-b promoter (18). Schimmel and colleagues have shown that the DNA sequence in the region upstream of the 5'-end of the mRNA is suggestive of two superimposed promoters that contain additional sequence elements known to regulate expression of other eukaryotic genes (32). The 5"region also has a remarkable homology to the overlapping promoters of the adenovirus EIIaE gene. By means of transient expression of the adenovirus E l a protein, they have been able to obtain enhanced expression of ck-b in an in vitro system (33). Hence, 1,25-(OH)zD3-induced differentiation could be associated with the expression of a factor (or factors) that enables complex formation on one of these enhancing elements, resulting in increased transcription. Studies are in progress to define the components of the two transcription complexes that form on the ck-b promoter following differentiation of ROS 17/2.8 cells.
In addition to transcriptional events, posttranscriptional regulation is also a factor in this system. There are several pieces of evidence that attest to the importance of posttranscriptional regulation in ck-b expression. Strauss and colleagues have shown that the 3"untranslated region of the ck-b mRNA is >70% conserved from chicken to man (20). This extraordinary level of conservation implies a functional role for a region of the mRNA that is generally associated with posttranscriptional regulation (34). We have described a system in the U927 monoblastic cell line that involves the 3'-untranslated region in the mediation of translational regulation of ck-b expression (19). Moreover, the involvement of multiple levels of regulation would be in keeping with the need for stringent regulation of expression to ensure appropriate subcellular distribution of energy. Mature osteoblasts require changes in cytoskeletal architecture in order to adhere to periosteal and endosteal bone surfaces and limit the exposure of underlying bone to osteoclasts. Creatine kinase activity has been shown to be closely associated with the cytoskeleton where the provision of ATP is needed for microtubular movement. Cytoskeletal changes and alterations in cell shape occur in osteoblasts following incubation with 1,25-(OH)zD3 (6). While transcriptional events effect an overall increase in ck-b mRNA level, posttranscriptional and translational mechanisms could be involved in targeting ck-b expression to appropriate subcellular compartments with increased energy needs following differentiation.
The data that implicate involvement of posttranscriptional regulatory mechanisms are the differences in the extent of enhancement of ck-b transcription, mRNA level, and protein abundance. ck-b mRNA and protein levels peak and reach steady state at 48 h of incubation with 1,25-(OH)2D3. At this point, the increase over untreated cells is 2.0-, 5.7-, and 2.1-fold for transcription rate, mRNA abundance and protein activity, respectively. The discrepancy between the increase in transcription rate and mRNA abundance indicates that mRNA stability is increased. In addition, as protein abundance is increased only 2.l-fold, not all of the increased mRNA is expressed as protein. Based on these data and our earlier finding that translational repression is an important factor in ck-b regulation (191, we hypothesize that a factor binds to the 3'untranslated region of the ck-b mRNA effecting both increased message stability and translational repression. Such a complex would explain the observation that mRNA abundance is increased to a greater extent than the increase in transcription rate and protein abundance. Supporting this hypothesis are the results of RNA mobility-shift analysis which show that a cytosolic factor in control and 1,25-(OH)2D3-treated ROS 17/2.8 cells interacts specifically with the 3"untranslated region of ck-b mRNA to produce a shifted band. No such shifted band is seen with a control RNA fragment that is similar in size but unrelated in sequence (derived from the 5'-region of ck-b mRNA). This interaction could also serve as a means for targeting the expression of ck-b to subcellular compartments. It is well established that ck-b activity is compartmentalized to cellular structures where energy is being actively expanded. In addition to the cytoskeleton (51, membrane pumps are other cellular structures with localization of ck-b activity (30). This is particularly relevant during development of osteoblasts as the differentiated cell has heightened activity of its membrane pumps to facilitate the deposition of extracellular matrix and mineralization. The mechanism by which this localization of expression is mediated remains unknown. Unlike other localized proteins, posttranslational processing is not a feature of ck-b expression. The suppression of ck-b mRNA translation, mediated by the interaction of its 3"untranslated region with a trans-acting cytosolic protein, is the probable means by which diffuse and indiscriminate expression is prevented. Completion of translation of the mRNA is prevented by this protein-mRNA complex at the 3'-end (19). In the micromilieu of specific subcellular compartments, local factors, such as pH and ADPIATP ratio, cause dissociation of the complex and completion of translation, resulting in increased local expression. Hentze and colleagues (35) have described a system in which cytosolic mRNAbinding proteins, present in excess, mediate translational regulation by changes in their affinity for the cis-acting element according to the presence or absence of local factors. Similarly, in Xenopus oocyte, a protein-RNA complex is formed on the 3"untranslated region of the vegetal mRNA which targets it to the appropriate subcellular compartment for expression (36).
These molecular mechanisms regulating ck-b expression have significant roles in osteoblast physiology. They are especially pertinent to the attainment of the mature phenotype which includes changes in cytoskeleton and increased activity of membrane pumps. An analogous situation would be the enhancement of creatine kinase expression during differentiation of muscle cells. MyoD, a transcription factor that is essential for the attainment of the differentiated muscle phenotype, specifically enhances creatine kinase expression by interacting with the "E" box on the promoter of the creatine kinase gene (12). It is probable that the trans-acting factors that mediate the enhancement of ck-b expression have additional roles in the overall process of osteoblast maturation. Inhibition of the upregulation of creatine kinase, with antisense RNA targeted to the ck-b mRNA, results in failure to attain the mature osteoblastic phenotype.2 The involvement of both transcriptional and posttranscriptional mechanisms in the regulation of ck-b is consistent with the need for stringent modulation, not only in terms of absolute quantities, but also in the targeting of expression to subcellular compartments involved in high energy utilization. The identification of cis-and trans-acting factors in this system will further enhance knowledge on the regulation of cellular energy metabolism in mature osteoblasts.


s2fieldsofstudy: Biology Medicine

text:
Changes in Eicosanoid and Tumour Necrosis Factor-α Production by Rat Peritoneal Macrophages During Carrageenin-Induced Peritonitis

Changes and correlations in cytokine and eicosanoid production by blood monocytes, non-purified and purified peritoneal cells during a carrageenin-induced peritonitis were investigated for a period of ten days. The cells were isolated and stimulated in vitro. Cytokine and eicosanoid production of the non-purified fraction increased steadily during peritonitis. During the whole episode of peritonitis the production capacity of granulocytes was very low and hardly any effect on the production capacity of macrophages (Mϕ) was observed. Cytokine and eicosanoid production of the non-purified fraction was mainly due to the presence of Mϕ. The production capacity of the peripheral blood monocytes was not similar to that of the peritoneal Mϕ.

Introduction
Inflammation is the reaction to an injury such as an invasion by infectious agents. The blood supply to the inflamed area increases, venular permeability increases and leucocytes migrate out of venules into the surrounding tissue. At the site of an acute inflammation polymorphonuclear granulocytes (PMNs) produce large amounts of reactive oxygen intermediates, release a variety of hydrolytic enzymes and phagocytose pathogens. By generating chemotactic factors, the migration of mononuclear cells (monocytes/M and lymphocytes) to the site of inflammation is stimulated. ',3 During the chronic phase of the inflammatory response, M, mononuclear phagocytes derived from blood monocytes, predominate over PMNs. In addition to acting as a nonspecific defence, activated M 5,6 will initiate and control the specific defence by presenting processed antigens to lymphocytes and by cytotoxity through direct cell-cell contact with tumour cells or infected cells, 8,9 during which they secrete cytokines and eicosanoids. 1,11 These inflammatory mediators also influence and regulate functions of other cells participating in the immune response, l'a Cytokines are proteins with regulatory functions. They can induce other cytokines with overlapping effects and form interactive networks with hormones and eicosanoids. M are a potent source of a wide variety of cytokines, 4 whereas granulocytes are reported to produce only a limited spectrum of cytokines. [15][16][17] ( 1994 Rapid Communications of Oxford Lid Eicosanoids are bioactive lipids derived from arachidonic acid (AA) by cyclooxygenase and lipoxygenase pathways. 8a9 Eicosa.noid production is species and tissue dependent, and often stimulus dependent. 2,21 The aim of this work was to study cytokine (tumour necrosis factor-m, TNF) and eicosanoid (leukotriene B4, LTB4; prostaglandin E2, PGE2; prostacycline, detected as 6-keto-prostaglandin Fire 6kPGF=; and thromboxane, TXB,) production capacity of peritoneal PMNs, M and blood monocytes after the induction of peritonitis with a carrageenin solution, a sulfated polygalactan which stimulates cell-mediated immunity. 22 It was of interest to determine whether, during an episode of peritonitis, changes occurred in (a) the differentiation of influxed cells; (b) the cytokine and eicosanoid production capacity of blood monocytes and peritoneal cells; (c) interactions between different types of peritoneal cells concerning their production capacity; and (d) if there was a correlation between the production of eicosanoids and TNF by blood monocytes and peritoneal M.

Materials and Methods
Animals and treatment: Young male Wistar rats (13 weeks, approximately 200 g, six rats per group) were injected intraperitoneally with 2 ml of a carrageenin solution (Marine Colloids Inc., USA, 1 mg/ml) on day 0. Six rats received an injection of saline (control group).
The peritoneal cavities, were washed twice with 20 ml PBS (pH 7.4, 4C, Oxoid, UK). The washings per rat were pooled, centrifuged (400 xg, 4C, 10 min) and suspended in 5 ml DMEM. 5 x 106 cells of this non-purified fraction were kept separate, the rest of this 'crude' fraction was separated on Percoll into a 'macrophage' and a 'granulocyte' fraction. These two purified fractions were washed three times with PBS (400 xg, 4C, 10 min), and suspended in DMEM (i x 106 cells/ml). A small sample of cells from each fraction was stained by Hemacolor (Merck, Germany) and the different cell types were counted using a microscope (Zeiss, standard 25, Germany). The viability of the cells was determined by trypan blue exclusion.
The cells were also incubated for 24 h in the absence or presence of lipopolysaccharide (LPS, 10 l.tg/ml final concentration in PBS, LPS from E. coli 0111:B4 in PBS (Sigma, USA), 37C, 7.5% COg). As a blank, PBS was added. At the end of the incubation the supernatant was centrifuged and kept at -80C until required for analysis.

Results
Concentration and differentiation of cells obtained from theperitoneal cavity. Induction of peritonitis in rats caused an increase of cells in the peritoneal cavity on day 1. The cell count dropped on the following days (Fig. 1A). A pronounced influx of granulocytes was seen on the first day which was followed by an influx of Me, resulting in a Me/ granulocyte ratio of 4:1 from day 3 to 10 ( Fig. 1B).
Cytokine and eicosanoid production by the crude fraction increased steadily during peritonitis. TNFc and TXB2 reached peak levels after 3 days, whereas PGE2 and 6kPGFI= reached a plateau 7 days after induction of the peritonitis. In comparison with levels of other eicosanoids the LTB4 production was low before and on the first day of the peritonitis and decreased significantly thereafter. By day 10 the production capacity returned to the initial level.
The production pattern of the cells from the macrophage fraction (Fig. 2B) was similar to that of cells from the crude fraction ( Fig. 2A). Inflammatory mediators were also produced by the granulocyte fraction (Fig. 2C), although the production capacity of these cells was very low in comparison with cells from the crude and macrophage fraction. The pattern of inflammatory mediator production of the granulocyte fraction did not significantly change in time. On the tenth day after induction of the peritonitis, the concentration of the cells in the granulocyte fraction was too low to permit in vitro incubations. The capacity of cells from the peripheral blood monocyte fraction to produce inflammatory mediators (Fig. 2D) was at least four times lower in comparison with both the crude and macrophage fractions. The production capacity of the cells from the monocyte fraction was about the same with or without stimulus. The patterns of the TNFz, PGE and 6kPGFI= production of cells from the monocyte fraction were different from those of the macrophage and crude fraction. After induction of peritonitis the TNF0t production of cells from the monocyte fraction increased significantly only on the tenth day, whereas PGE2 and TXB,. reached their highest level on the third day. The 6kPGFlu level did not change during peritonitis. The LTB4 production was negligible.
Correlations between inflammatory mediators produced in the same fraction: In the crude fraction ( Fig.   2A) there was a significant positive correlation between the production of PGE,. and 6kPGFI= and PGE2 and TXB,. (r= 0.6365 and r= 0.7145). Similar significant correlations were found in the macrophage fraction (Fig. 2B, r= 0.5580 and r= 0.6106). In the monocyte fraction (Fig. 2D) the only significant positive correlation between the inflammatory mediators was between PGE and WXB (r= 0.8881).
Correlation between inflammatory mediators in different fractions: In Fig. 2A and 2B the patterns of inflammatory mediators produced by the stimulated crude and macrophage cell fractions are presented. There was always a significant positive correlation between these two fractions. The correlation varied from r= 0.5671 for LTB to r 0.8586 for TNFo:. (PGE,., r= 0.6254; 6kPGFI=, r= 0.7536; and TXB2, r= 0.7502). This correlation remained significant when we assumed that production of the cell inflammation mediators in the crude and macrophage fraction were only derived from the Me (calculated to 100% macrophages, LTB4=0.4649; PGE2 0.5855; 6kPGFI= 0.5506; TXB2 0.7810; and TNFo: 0.8674).
There were, however, some significant differences between the calculated (to 100% Me) production levels of the crude and macrophage fractions. When separate days were considered the production levels of the calculated crude fraction in comparison with the calculated macrophage fraction was on day 1 lower for PGE2 and 6kPGF=, and on day 3 higher for gWB4, TXB2 and TNFo: (Fig. 3 A-E).
Correlation coefficient between inflammatory mediators from monocyte and macrophage fraction: Only the basal TNFz production of the cells from the monocyte and macrophage fraction were clearly correlated (r= 0.5421).

Discussion
Characterization of inflammatory mediators produced by granulocytes and monocytes/macrophages during induced inflammation in animal models, may be helpful to the understanding of inflammatory diseases and their treatment. The present study investigated the changes and correlations in cytokine and eicosanoid production capacity of peripheral blood monocytes, non-purified ('crude' fraction) and purified ('macrophage' and 'granulocyte' fraction) peritoneal cells during a carrageenin-induced peritonitis in rats.
In the present study, where an influx of leucocytes into the peritoneum was achieved after carrageenin injection, the number of PMNs increased in the acute phase (day 1) and thereafter decreased quickly. On day 0, before inducing the peritonitis, there was a reasonable number of Me present in the peritoneal cavity. The number of Me increased from day 1 until 7 days after induction of the peritonitis, resulting in a mainly Me cell population. Previous histological studies had similar results, '4,25 although a quantitative and kinetic method showed that both monocytes and PMNs from rat migrate rapidly to the inflamed site, the migration of PMNs, however, also declined long before the migration of monocytes started to decrease. The pattern of this migration can be explained with the function of the leucocytes in inflammation. In the initial phase both PMNs and Me are involved in nonspecific defence mechanisms. ,2 In addition, Me will also initiate and control the specific defence.  The mechanism of this selective migration is not clear. Leucocyte adhesion to the blood vessel endothelium, which is followed by transendothelial migration, is a multistep process in which several adhesion molecules are involved, z6,z7 Our experiment showed that the inflammatory mediator production capacity of the non-purified fraction, which was harvested from the inflamed peritoneal cavity, changed with time. The production of inflammatory mediators in the non-purified fraction was mainly caused by the presence of M) during carrageenin-induced peritonitis. This is, first of all, based on the significant correlation between the eicosanoid and TNFot production capacity of the macrophage fraction and the crude fraction, these correlations hardly changed when it was assumed that these fractions only contained M. Secondly, the production capacity of the granulocytes appeared to Changes in eicosanoid and TNF production be very low. Moreover the production capacity of the granulocyte fraction did not correlate with the crude fraction.
When the production levels of the non-purified fraction was compared with the macrophage fraction, assuming that the inflammatory mediators were only produced by M, it was observed that the production of PGE,. and 6kPGFI= had decreased on day I and the production of LTB4, TXB and TNF had increased on day 3. The results of these effects could be an amplification of the inflammation, due to the decrease of anti-inflammatory substances and the subsequent increase of pro-inflammatory substances.
The low production capacity of the crude fraction in the acute phase followed by an increasing production capacity in the chronic phase could also be explained by presence of the M. Blood monocytes and resident M transform from primed into active M at the side of the inflammation, after which profound changes occur in morphology and function of these cells. The M present at day 1 in the peritoneal cavity, probably were not primed in vivo at this time, which could be the reason that the eicosanoid and cytokine production capacity of these M were so low after stimulation in vitro with LPS. Once these M were primed in vivo, also the new monocytes that migrate into the peritoneal cavity, the production capacity of these monocytes/M steadily increased, showing in vitro a differentiated pattern for the eicosanoids and TNF. This pattern probably depends on the function of the metabolite.
As peripheral blood monocytes transform into peritoneal m, the correlation between these cells was investigated. The eicosanoid and TNF production capacity of blood monocytes was not similar to that of M during peritonitis. It was clearly shown that through influx and transformation of monocytes into M the synthesis of mediators completely changed.
In conclusion, M are the main source of the inflammatory mediators whose production is dependent on the episode of the carrageenin-induced peritonitis.Characterization of purified M in vitro in animal and human models will be helpful to the understanding of inflammatory diseases and their treatment. Peripheral blood monocytes do not reflect mediator production of M present at the inflammatory site.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Activation of the Mitogen-activated Protein Kinase Pathway by the Erythropoietin Receptor*

The erythropoietin receptor (EpoR) belongs to the cy- tokine receptor family, members of which lack a tyrosine kinase domain. Recent studies, however, have shown that a cytoplasmic tyrosine kinase, JAK2, inter-acts with the cytoplasmic domain of the EpoR and becomes activated upon binding of Epo to the receptor. Epo has also been shown to stimulate activation of Ras and Raf-1. The present studies were undertaken to ex- amine the possible involvement of Epo-induced tyrosine phosphorylation in activation of the Radmitogen-acti-vated protein kinase ( M A P kinase) pathway and to de- termine its significance on the growth signaling from the EpoR. In an interleukin (IL)-3-dependent cell line expressing the transfected wild-type EpoR, Epo, or IL-3 induced tyrosine phosphorylation of Shc and its associa- tion with Grb2. These cytokines also induced tyrosine phosphorylation and activation of MAP kinase isoforms ERKl and E m . A mutant EpoR with a carboxyl-termi-nal deletion of 108 amino acids (H mutant), which is mitogenically functional but lacks tyrosine phosphoryl- ation sites in the carboxyl-terminal region, showed

The erythropoietin receptor (EpoR) belongs to the cytokine receptor family, members of which lack a tyrosine kinase domain. Recent studies, however, have shown that a cytoplasmic tyrosine kinase, JAK2, interacts with the cytoplasmic domain of the EpoR and becomes activated upon binding of Epo to the receptor. Epo has also been shown to stimulate activation of Ras and Raf-1. The present studies were undertaken to examine the possible involvement of Epo-induced tyrosine phosphorylation in activation of the Radmitogen-activated protein kinase ( M A P kinase) pathway and to determine its significance on the growth signaling from the EpoR. In an interleukin (IL)-3-dependent cell line expressing the transfected wild-type EpoR, Epo, or IL-3 induced tyrosine phosphorylation of Shc and its association with Grb2. These cytokines also induced tyrosine phosphorylation and activation of MAP kinase isoforms ERKl and E m . A mutant EpoR with a carboxyl-terminal deletion of 108 amino acids (H mutant), which is mitogenically functional but lacks tyrosine phosphorylation sites in the carboxyl-terminal region, showed markedly diminished abilities to induce tyrosine phosphorylation of Shc and to phosphorylate and activate MAP kinases. A mutant receptor (PM4 mutant) inactivated by a point mutation, T r p 2 * 2 to Arg, which abrogates the interaction with JAK2, failed to induce any effect on Shc or MAP kinases. In cells expressing a mutant EpoR that is constitutively activated by a point mutation, Arg12' to Cys, in the extracellular portion of the receptor, neither tyrosine phosphorylation of Shc nor activation of MAP kinases by phosphorylation was detectable without stimulation with Epo or IL-3. These results suggest that the carboxyl-terminal region of EpoR may play a crucial role in activation of MAP kinases through the Ras signaling pathway which may be activated by tyrosine phosphorylation of Shc and its association with Grb2. The activation of MAP kinases, however, failed to correlate with the mitogenic activity of mutant EpoRs and thus may not be required for growth signaling from the EpoR.
Erythropoietin (Epo)' is a hematopoietic growth factor that regulates the growth and differentiation of erythroid progeni-* This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan, from the Uehara Memorial Foundation, and from the Yamanouch1 Foundation for Research on Metabolic Disorders. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed. Tel.: 81-3-3813-6111 (ext. 3218); Fax: 81-3-3818-0448. The abbreviations used are: Epo, erythropoietin; EpoR, erythropoietin receptor; IL, interleukin; SH2, Src homology 2; SH3, Src homology tor cells (1,2). The receptor for Epo (EpoR) (3,4) belongs to the cytokine receptor family which includes receptors for most of the hematopoietic growth factors (5, 6). The members of this family characteristically have 4 positionally conserved cysteines and a Trp-Ser-X-TlpSer (WSXWS) motif in the extracellular domain. Except for a short region showing a limited sequence similarity (7-101, the intracellular region of these receptors is quite diverse and lacks any motifs that would indicate potential function, such as the tyrosine kinase domain. Most of the hematopoietic growth factors, however, induce tyrosine phosphorylation of cellular proteins (3,6). Furthermore, introduction of a variety of activated tyrosine kinases has been shown t o abrogate dependence of hematopoietic cell lines on these hematopoietic growth factors (11). These observations have led to a hypothesis that tyrosine phosphorylation plays a critical role in the growth signaling from the hematopoietic cytokine receptors.
Using Epo-dependent cell lines expressing the endogenous or transfected EpoR, we and others have shown that Epo rapidly induces tyrosine phosphorylation of a series of cellular substrates, some of which are also phosphorylated by stimulation with interleukin (1L)-3 (12, 13). We further revealed that a membrane proximal cytoplasmic region, showing a limited homology with the other cytokine receptors, is critical for induction of tyrosine phosphorylation, the expression of a series of immediate-early genes, including c-myc, c-fos, and egr-1, and mitogenesis (10). Upon Epo binding, the carboxyl-terminal region of the EpoR becomes phosphorylated on tyrosine (13,14) and physically associates with the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase (15) but is not required for growth signaling (13,16,17). Very recently, we have revealed that Epo stimulation induces binding of JAK2, a member of the J A K family of cytoplasmic tyrosine kinases, to the functionally critical membrane proximal region of the receptor and activates its kinase activity (18,19). The JAK2 kinase was thus suggested to couple Epo binding to tyrosine phosphorylation. However, little has been known how the signal is transmitted to the downstream signaling elements.
For both receptor and cytoplasmic tyrosine kinases, activation of Ras has been regarded as a critical step in inducing mitogenesis or differentiation (20,211. The proteins Grb2, Shc, and mSosl have been implicated in the control of Ras by tyrosine kinases in a number of different biological systems. Grb2 is an adapter protein that lacks a catalytic domain and is composed of one Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains (22,23). Grb2 binds to a subset of 3; MAP kinases, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK-activating kinase; MBP, myelin basic protein; GM-CSF, granulocyte-macrophage colonystimulating factor; G-CSF, granulocyte colony-stimulating factor; PAGE, polyacrylamide gel electrophoresis. autophosphorylated receptor tyrosine kinases through its SH2 domain (22,231 and simultaneously associates through its SH3 domains with mSosl, a guanine nucleotide-releasing protein that activates Ras by inducing exchange of GDP for GTP on Ras (24). Grb2 thus repositions mSosl adjacent to Ras, which is located at the plasma membrane. The shc gene encodes three overlapping proteins of 46, 52, and 66 kDa which possess a carboxyl-terminal SH2 domain and a glycine/proline-rich region but no obvious catalytic domain (25). Shc proteins become tyrosine-phosphorylated upon activation of a variety of receptor tyrosine kinases (25-27) and are phosphorylated constitutively in cells transformed by the v-Src or v-Fps tyrosine kinases (28). Shc has been implicated in activation of Ras, because the tyrosine-phosphorylated Shc proteins associate with Grb2 and mSosl(26,29,30). Moreover, overexpression of Shc proteins led to Ras-dependent neurite outgrowth in PC12 cells (26).

A B
Recent studies have revealed that tyrosine kinase pathway activates mitogen-activated protein kinases ( M A P kinases), also known as extracellular signal-regulated kinases (ERKs), in a Ras-dependent manner (31, 32). MAP kinases are serine/ threonine kinases that phosphorylate well studied regulatory proteins, including transcription factors, membrane proteins, cytoskeletal elements, and other protein kinases (31, 33). MAP kinases are thus thought to be key intermediate regulatory proteins functioning in signal transduction networks. MAP kinases achieve maximum activity when phosphorylated on both tyrosine and threonine residues by MAP kinase/ERK-activating kinase (MEK), a dual specificity kinase (31, 32). MEK in turn is phosphorylated and thus activated by a serine/ threonine kinase, 32). It is known that activated Ras physically associates with Raf-1 and that phosphorylation is required for activation of Raf-1 (32). However, the mechanism how Ras activates Raf-1 remains to be known.
The cascade of intracellular signal transducing events leading to activation of MAP kinases may also be involved in the EpoR signaling, because Epo also activates Ras (34, 35) and induces phosphorylation and activation of Raf-1 (36). The present studies were undertaken to examine the involvement of Ras-activating proteins and MAP kinases in the EpoR signaling. Epo was found to induce tyrosine phosphorylation of Shc and its association with Grb2 in Epo-stimulated cells. Epo also induced tyrosine phosphorylation and activation of MAP kinases. Among mutant EpoRs, the ability to induce phosphorylation of Shc and its association with Grb2 correlated with the ability to activate MAP kinases. However, activation of MAP kinases failed to correlate with the mitogenic activity of mutant EpoRs.

MATERIALS AND METHODS
Cells and Reagents-A clone of 32D cells, an IL-3-dependent cell line originally isolated from long term bone marrow cultures, has been previously described (37). 32D clones expressing the wild-type or various mutant EpoRs and IL-3-dependent DA3 cells expressing the wild-type EpoR were also described previously (10, 13) and maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and 10% WEHI-3 conditioned medium as a source of IL-3.
An expression plasmid for activated mutant EpoR with an ArglZ9 to Cys mutation was constructed by primer-mediated mutagenesis using the polymerase chain reaction method as described previously (10). Transfection of the plasmid into DA3 cells and isolation of a clone expressing the mutant EpoR were also carried out as described previously (13). In brief, DA3 cells were transfected with 10 pg of the expression plasmid and 1 pg of the pSV2neo plasmid by electroporation and selected in medium containing G418. Six clones were isolated by limiting dilution and confirmed to grow in medium containing neither IL-3 nor Epo. These clones were then analyzed by "'I-Epo binding assays, and the clone that bound the highest radioactivity was used for the subsequent studies.
For measurement of the cell number increase, cells were cultured in 25-cm2 culture flasks a t a density of 1 x 105/ml (5 ml of culture) in 10% fetal calf serum-containing RPMI 1640 medium supplemented with 10% WEHI-3 conditioned medium or with 4 unitdml human recombinant Epo. Culture media were changed every 2 days. Viable cell counts were determined by trypan blue staining.
The preparation and properties of rabbit polyclonal antisera against the cytoplasmic portion of recombinant murine EpoR (38) or against synthetic peptides from JAKl and JAK2 (39) have been described. Antiphosphotyrosine monoclonal antibody (4G10) and rabbit antisera against Shc and ERKl (erkl-CT) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). A rabbit antiserum against ERK2 and a monoclonal antibody against Grb2 were purchased from Transduction Laboratories (Lexington, KY). Recombinant human Epo was kindly provided by Sankyo Pharmaceutical Co. Ltd. (Tokyo, Japan).
Immunoprecipitation and Immunohlotting-For stimulation with Epo or IL-3, cells were starved for 12 h without IL-3 in complete medium. The cells were then left unstimulated as a negative control or stimulated with a saturating concentration of Epo or IL-3 a t 37 "C for 10 min. The cells were lysed in a lysis buffer containing 1% Triton X-100, 20 mM Tris-HCI (pH 7.5), 150 mhl NaCI, 1 mM EDTA, 100 PM sodium orthovanadate, 1 m M phenylmethylsulfonyl fluoride, and 10 pg/ml each of aprotinin and leupeptin. For immunoprecipitation of MAP kinases, 1 x 10' cells were lysed in 100 p1 of boiling lysis buffer containing 1% SDS and 10 mM Tris-HCI (pH 7.4) and boiled for another 5 min. The lysate was then diluted 10-fold with the Triton X-100 lysis buffer and denatured MAP kinases were immunoprecipitated with anti-MAP kinase antibodies.
For immunoprecipitation, a relevant antibody was added to the lysates along with protein A-Sepharose beads and incubated for 4 h a t 4 "C. The beads were washed extensively, and the proteins bound to the beads were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, immunoblotted with the indicated antibody, and developed by the enhanced chemiluminescence (ECL) system (Amersham Corp.). Aliquots of the cell lysates were also subjected to immunoblotting after directly mixed with equal volumes of 2 x Laemmli's sample buffer and heated a t 100 "C for 5 min. For reprobing with a different antibody, the membranes were treated at 50 "C for 30 min with stripping buffer containing 100 mh1 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HC1 (pH 6.7).
For determination of the stoichiometry of tyrosine phosphorylation of Shc and MAP kinases, 2 x 10' cells stimulated with 75 unitdm1 Epo for 10 min were lysed in 1 ml of the lysis buffer. After clarification, 50 pl of the lysate was mixed with the equal volume of 2 x SDS buffer and boiled for 5 min, whereas 500 pl of the lysate was immunoprecipitated with 10 pg of the anti-phosphotyrosine 4G10 antibody conjugated with agarose beads and subsequently eluted with 50 pl of 1 x SDS buffer by boiling for 5 min. The whole cell lysate and the immunoprecipitate were diluted -E p l U

200-
appropriately and varying amounts of the samples were subjected to immunoblot analysis using antibody against phosphotyrosine, Shc, or MAP kinases.
Kinase Assays of Anti-MAP Kinase Immunoprecipitates in Myelin Basic Protein fMBP)-containing Gels after SDS-PAGE-Determination of MAP kinase activity in MBP-containing gel was carried out essentially as described (40). In brief, anti-ERK1 immunoprecipitates were prepared as described above and electrophoresed on an SDS-12% polyacrylamide gel containing 0.5 mg/ml MBP. After removing SDS with buffer containing 20% 2-propano1, the gel was denatured with 6 M guanidine HCI and then renatured in a 0.04% Tween 40-containing buffer. Phosphorylation of MBP was carried out by incubating the gel a t 22 "C for 1 h in 40 mM HEPES (pH 7.51, 0.1 mM EGTA, 20 mM MgCI,, 20 p~ ATP, and 25 pCi of [Y-:'~P]ATP. After incubation, the gel was washed extensively with 5% trichloroacetic acid, 1% pyrophosphate solution, dried, and subjected to autoradiography. The radioactivity of phosphorylated MBP was also quantified by Bio-Imaging Analyzer BAS2000 (Fuji Film, Tokyo).

RESULTS
Epo Induces Tyrosine Phosphorylation of Shc and Its Association with Grb2-To explore the signal transduction pathways from the EpoR, we first examined whether Epo stimulation induces tyrosine phosphorylation of Shc, which has been implicated in coupling receptor tyrosine kinases to the Ras signaling pathways. As shown in Fig. lA, Epo stimulation induced tyrosine phosphorylation of 150-, 130-, 97-, 92-, 72-, 70-, 52-, and 49-kDa proteins in the 32D cells expressing the wildtype EpoR (32DlEpoR-Wt). IL-3 stimulation induced an almost identical pattern of tyrosine phosphorylation except that the 72-kDa protein was not phosphorylated after IL-3 stimulation. In accordance with our previous results (13,18,19), anti-phosphotyrosine blotting of immunoprecipitates obtained with relevant antisera showed that the 72-and 130-kDa substrates were the EpoR and JAK2, respectively, whereas it was revealed that JAKl was not phosphorylated after stimulation with these cytokines in the 32D cells examined.
Anti 49-kDa proteins in cells stimulated with Epo or IL-3 (Fig. lA ).
These proteins were identified as Shc by reprobing with anti-Shc (Fig. 1B). In addition, tyrosine phosphorylated 210-and 150-kDa proteins were coimmunoprecipitated with Shc after Epo or IL-3 stimulation.
To address the functional significance of tyrosine phosphorylation of Shc, we next examined previously characterized 32D clones expressing various mutant EpoRs (10, 13). 32DEpoR-H cells express the truncated H mutant lacking the carboxylterminal 108 amino acids. Although this mutant lacks the tyrosine phosphorylation sites in the carboxyl-terminal region and fails to associate with PI 3-kinase (151, it retains the abilities to activate JAK2 (18,19) and to transduce a mitogenic signal (10, 13). The mitogenic response of 32D/EpoR-H, which was selected by the G418 resistance, to Epo was thus comparable with that to IL-3 as determined by ["]thymidine incorporation (10) or by the growth curves (Fig. 2). 32DEpoR-PM4 cells express the PM4 mutant, which contains a mutation, Trp2*' to Arg (W282R), in a membrane proximal region of the cytoplasmic domain that shows homology with other members of the cytokine receptor superfamily (10). Having lost the ability to associates with JAK2 to activate its kinase activity (19), the PM4 mutant fails to induce tyrosine phosphorylation of cellular substrates, including the receptor itself, and to elicit a mitogenic response (10, 13). 32DEpoR-PM4, thus, failed to grow in response to Epo, as shown in Fig. 2.
The abilities of these mutant EpoRs to induce tyrosine phosphorylation of cellular proteins were first examined by antiphosphotyrosine blotting of total cell lysates. As shown in Fig.  3A, Epo induced tyrosine phosphorylation of p130 (JAK2) and p92 in 32DEpoR-H cells. In most of the repeated experiments, the tyrosine phosphorylation of JAK2 in these cells was enhanced as compared with that in the 32DEpoR-Wt cells, whereas p70 showed variable degrees of tyrosine phosphorylation in response to Epo. On the other hand, the tyrosine phosphorylation of p52 and p49, which correspond to Shc, after Epo stimulation in 32DEpoR-H was constantly and remarkably diminished as compared with that observed after IL-3 stimulation in this cell line or after Epo stimulation in 32DEpoR-Wt. The PM4 mutant failed to induce any detectable tyrosine phosphorylation of cellular substrates in response to Epo (Fig. 3A).
The tyrosine phosphorylation of Shc in these cell lines was then directly examined by anti-phosphotyrosine blotting of anti-Shc immunoprecipitates. Results shown in Fig. 3B confirmed that the Epo-induced tyrosine phosphorylation of Shc was severely impaired or abolished in 32DEpoR-H or 32D/ EpoR-PM4, respectively.
Because tyrosine-phosphorylated Shc has been shown to activate the Ras signaling pathway by physically associating with the Grb2-mSosl complex through the SH2 domain of Grb2, the anti-Shc immunoprecipitates were reprobed with anti-Grb2 to examine the association of Grb2 with Shc in Epo-stimulated cells. As shown in Fig. 30, in 32DEpoR-Wt, Grb2 was shown to coimmunoprecipitate with Shc after stimulation with Epo or IL-3. 32DEpoR-H cells also showed the Epo-induced association of Grb2 with Shc. However, the amount of Grb2 associated with Shc was markedly decreased in accordance with the decrease in tyrosine phosphorylation of Shc. Epo failed to induce the association of Grb2 with Shc in 32DEpoR-PM4 (Fig. 30).

nrosine Phosphorylation and Activation of MAP Kinases
Induced by Epo Stimulation-Recent studies have revealed that activation of MAP kinases by a variety of receptor tyrosine kinases is mediated by Ras (31,32). Since Epo has been shown to stimulate the activation of Ras (34, 35), we next examined whether Epo stimulation induces tyrosine phosphorylation and activation of MAP kinases, which are activated by phosphoryl- ation on both tyrosine and serinekhreonine residues (31, 32). To examine tyrosine phosphorylation of MAP kinases, immunoprecipitates obtained with anti-ERK1 (erk 1-CT), which also recognizes ERK2, were examined by anti-phosphotyrosine blotting. As shown in Fig. 4A, 42-and 44-kDa species of MAP kinases, which should correspond to ERK2 and ERK1, respectively, were found to be tyrosine-phosphorylated after Epo or IL-3 stimulation in 32DEpoR-Wt. However, tyrosine phosphorylation of MAP kinases induced by Epo stimulation was remarkably reduced in 32DEpoR-H (Fig. 4A). Reprobing of the membrane with anti-ERK1 confirmed that the 42-and 44-kDa species of tyrosine-phosphorylated proteins are directly recognized by anti-ERK1 and demonstrated equal loading of samples (Fig. 4A, center panel). The anti-ERK1 immunoprecipitates were then subjected to the MAP kinase assay in MBP-  containing gel. As shown in Fig. 4A (lower panel), Epo stimulation was found to stimulate the kinase activities of both ERKl and ERK2. However, although the effect of Epo on the MAP kinase activity was about half as much as that of IL-3 in 32DIEpoR-Wt, Epo showed only a marginal effect on the MAP kinase activity in 32DIEpoR-H (Fig. 4, A and B ). In 32DIEpoR-PM4 cells, Epo failed to induce any detectable tyrosine phosphorylation or activation of MAP kinases (data not shown). Stoichiometry of the Tyrosine Phosphorylation of Shc and MAP Kinases-We next tried to determine what fraction of Shc or MAP kinases is tyrosine-phosphorylated in response to Epo stimulation. For this purpose, phosphotyrosyl proteins were immunoprecipitated with the 4G10 antibody from Epo-stimulated 32DIEpoR-Wt cell lysate and, along with varying amounts of whole cell lysate, subjected to immunoblot analyses. First, the efficiency of immunoprecipitation with 4G10 was evaluated by immunoblotting with this antibody. The amount of tyrosine-phosphorylated Shc or MAP kinases in the 4G10 immunoprecipitate was then estimated by immunoblotting with anti-Shc or anti-MAP kinase, respectively. As shown in Fig. 5, the efficiency of immunoprecipitation with 4G10 was rather low and varied significantly with each phosphotyrosyl proteins, which may be a t least partly because some phosphorylated tyrosine residues are involved in intramolecular or intermolecular interaction with the SH2 domains and thus may not bind with 4G10. Densitometric analysis of the results shown in Fig. 5, representative of three repeated experiments, revealed that the efficiency of 4G10 immunoprecipitation of tyrosine-phosphorylated Shc or ERK-1 was -8 or -5%, respectively, whereas -3 or -0.5% of the total cellular Shc or ERK-1, respectively, was shown to be present in the 4G10 immunoprecipitates. From these results, it was calculated that -38 or -10% of total cellular Shc or ERK-1, respectively, undergoes tyrosine phosphorylation in response to Epo in 32DIEpoR-Wt cells. Thus, Epo induced significant but not near-stoichiometric tyrosine phosphorylation of Shc and MAP kinases. The rather low stoichiometry of tyrosine phosphorylation, particularly of MAP kinases, is compatible with our results that only a barely detectable activity of MAP kinases was observed in Epo-stimulated 32DEpoR-H cells, in which Epo-induced tyrosine phos- phorylation of Shc and MAP kinases is markedly diminished as compared with that in 32DIEpoR-Wt.

Cells Expressing a Constitutively Activated EpoR Mutant Show Epo-independent Growth without Tyrosine Phosphorylation of Shc or Activation of MAP
Kinases-Previously, a single point mutation, resulting in an Arg to Cys change a t residue 129 of the extracellular domain of EpoR, has been shown to activate the receptor independent of Epo binding (41,421. The R129C mutant was confirmed to abrogate the factor-dependence of DA3 cells, because six subclones of transfectants, selected by the G418 resistance due to the cotransfected pSV2neo plasmid, grew in the absence of IL-3 or Epo; the clone used in this study grew comparably in medium with or without Epo (Fig. 6). We examined the tyrosine phosphorylation states of Shc and MAP kinases as well as the activity of MAP kinases in a DA3 clone that expresses this mutant receptor and thus grows without added growth factors. As shown in Fig. 7, antiphosphotyrosine and anti-Grb2 blotting of anti-Shc immunoprecipitates showed that tyrosine phosphorylation of Shc and its association with Grb2 was dependent on Epo stimulation in these cells showing the Epo-independent growth. Tyrosine phosphorylation and activation of MAP kinases were also dependent on stimulation with Epo or IL-3 (Fig. 8). These cells were thus found to grow without showing tyrosine phosphorylation of Shc or activation of MAP kinases in growth factordeficient medium.

DISCUSSION
The present studies demonstrated that Shc and MAP kinases are among the substrates of tyrosine phosphorylation induced by Epo stimulation. In addition, Epo induced physical association of Shc with Grb2 and activated the catalytic activity of MAP kinases. However, Epo did not have any effect on these signaling molecules in cells expressing the PM4 mutant EpoR, which has a point mutation, Trp282 to Arg, abolishing the ability of EpoR to couple with JAK2 to transduce a mitogenic signal. The effects of Epo on Shc and MAP kinases were markedly diminished in cells expressing the mitogenically functional, truncated H mutant, which has lost tyrosine phosphorylation sites in the carboxyl-terminal region. In cells expressing the mutant EpoR that is constitutively activated by a point mutation, Arg'" to Cys, tyrosine phosphorylation of Shc and its association with Grb2 as well as tyrosine phosphorylation and activation of MAP kinases were not observed without stimula- examined. A tyrosine-phosphorylated protein of 145 kDa (43,44) or 140 kDa (45), which should correspond to the 15O-kDa tion with Epo or IL-3. Thus, in cells expressing these mutant protein in the present studies, were also found to be associated EpoRs, activation of MAP kinases correlated with tyrosine with Shc in cells stimulated with Epo or IL-3. Although the phosphorylation of Shc and its association with Grb2. Taken identity of this species remains unknown, we found in a previtogether with previous reports showing that Epo activates Ras ous study that this 150-kDa protein coimmunoprecipitated and Raf-1 (34-361, the present results suggest that Shc and with the EpoR or JAK2 in digitonin lysates and underwent Grb2 may mediate activation of the Ras/MAP kinases pathway tyrosine phosphorylation in vitro (19). It is thus possible that from the EpoR and that the carboxyl-terminal region of the the EpoR-JAK2 complex physically associates with the 140receptor may play a major role in activation of this pathway. kDa protein, Shc, and Grb2, although the association may be Activation of MAP kinases, however, may not be required for the transduction of growth signal from the EpoR, because the activity of MAP kinases did not correlate with proliferation of cells expressing the mutant EpoRs.
Recent studies also demonstrated that Epo or IL-3 induces tyrosine phosphorylation of Shc and its association with Grb2 (43)(44)(45). Damen et al. (43) also observed that tyrosine-phosphorylated Shc associates with the EpoR in a human M07 cell line expressing transfected murine EpoR. However, association of transient or unstable.
Although the mechanism of how the EpoR activates Ras has remained elusive, inhibition of the Epo-induced activation of Ras by tyrosine kinase inhibitors suggested that it may be mediated through tyrosine phosphorylation (34). In a previous study (341, GTPase-activating protein was implicated in the Epo-induced activation of Ras, because Epo induced tyrosine phosphorylation of Ras GTPase-activating protein in a human erythroleukemia cell line (HEL). On the other hand, it was Shc with the EpoR was not observed in DA3 transfectants in demonstrated in the present study and also in previous studies their studies (43). We also failed to observe any significant (43)(44)(45) that Epo stimulation induces tyrosine phosphorylation association of the EpoR with Shc in the 32D or DA3 clones of Shc and its association with Grb2. Since Shc is strongly implicated in the activation of Ras through its interaction with Grb2 and indirectly with mSos1 (24, 26), Epo-induced activation of Ras may be mediated through tyrosine phosphorylation of Shc. In accordance with this idea, in cells expressing the mutant EpoRs, tyrosine phosphorylation of Shc and its association with Grb2 correlated with activation of MAP kinases, which are major targets of the Ras signaling pathway (31, 32). Very recently, we found that Epo also induces tyrosine phosphorylation of Vav (46), which has also been implicated in the Ras signaling pathway (47). However, the tyrosine phosphorylation of Vav induced by Epo did not show correlation with the activation of MAP kinases in cells expressing the various mutant EpoRs (46). Therefore, a role, if any, of Vav in the Epoinduced activation of Ras remains to be known.
Recent studies have demonstrated that activation of MAP kinases is involved in signaling from most of the members of cytokine receptor family. However, the functional significance of MAP kinase activation in regulation of growth and differentiation of hematopoietic cells by these cytokines remains elusive. In fibroblast, the MAP kinase activity was shown to be required for proliferation (48). Correlation of proliferative response with activation of MAP kinases has also been shown in hematopoietic cells stimulated with G-CSF; G-CSF activates MAP kinases in cell lines that proliferate in response to G-CSF, whereas neither G-CSF-induced granulocytic differentiation of 32D cells nor nonproliferative response of mature neutrophils to G-CSF was associated with MAP kinase activation (49). On the other hand, GM-CSF induces tyrosine phosphorylation and activation of MAP kinases in nonproliferative mature neutrophils (50,51). Dissociation between mitogenesis and MAP kinase activation has also been demonstrated in hematopoietic cell lines that proliferate in response to IL-4 without activation of MAP kinases (52). In the present studies, activation of MAP kinases also failed to correlate with mitogenesis, since the truncated H mutant, whose mitogenic function is intact, showed a significant impairment in activation of MAP kinases, whereas cells expressing the constitutively activated EpoR mutant grew in growth factor-deficient medium without showing any detectable MAP kinase activation. These observations raise a possibility that activation of MAP kinases may play a role in transduction of signals other than a mitogenic signal.
The present studies indicated that the membrane distal cytoplasmic region of the EpoR may play a major role in tyrosine phosphorylation of Shc and activation of MAP kinases. Sat0 et a2. (53) similarly reported that the membrane distal cytoplasmic region of the common p subunit of the GM-CSF receptor is essential for induction of tyrosine phosphorylation of Shc and for activation of Ras, Raf-1, and MAP kinases. This region of the receptor was also required for induction of c-fos and c-jun by GM-CSF stimulation. The authors thus suggested that GM-CSF-induced tyrosine phosphorylation of Shc may cause activation of Ras, Raf-1, and MAP kinases and ultimately lead to the expression of c-fos and c-jun. Recent studies have also shown that MAP kinases activate the transcription of c-fos by phosphorylating, directly or indirectly, factors that bind the serum-responsive element in the promoter region of the c-fos gene (31,33). On the other hand, a previous study showed that induction of c-fos expression by Epo stimulation was not impaired in cells expressing the truncated H mutant, which is defective in activating the MAP kinase pathway (10). Intriguing in this regard is a recent study disclosing Epo-induced tyrosine phosphorylation and activation of 91and 84-kDa proteins that translocate from the cytoplasm to the nucleus and form DNA-binding complexes which recognize the sis-inducible element in the c-fos promoter (54). A similar Ras-independent signaling pathway directly activating DNA-binding proteins that recognize the sis-inducible element is also utilized by receptors for various growth factors, cytokines, and interferons (55,56). Importantly, the J A K family of tyrosine kinases, which includes JAK2, is strongly implicated in this pathway (55,561. It is thus tempting to speculate that JAK2, which is activated by the H mutant, may activate a latent transcription factor in the cytoplasm through tyrosine phosphorylation and thus leads to activation of the promoters of the c-fos gene and other genes involved in cellular proliferation.