extfieldsofstudy: Biology Medicine

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Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase

We have developed a novel technique with which to investigate the morphological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytochemistry to outline traffic into and out of the Golgi apparatus at the electron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secretory pathway and the activity is efficiently cleared from cells. Investigation of the morphological elements involved in the itinerary of soluble ER proteins using ssHRP tagged with the ER retention motif (ssHRPKDEL) shows that it progresses through the Golgi stack no further than the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRPKDEL occurs via vesicular carriers as well as by tubular elements. ssHRP has also been used to investigate the trans side of the Golgi complex, where incubation at reduced temperatures outlines the trans-Golgi network with HRP reaction product. Tracing the endosomal compartment with transferrin receptor in double-labeling experiments with ssHRP fails to show any overlap between these two compartments.

Abstract. We have developed a novel technique with which to investigate the morphological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytochemistry to outline traffic into and out of the Golgi apparatus at the electron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secretory pathway and the activity is efficiently cleared from cells.
Investigation of the morphological elements involved in the itinerary of soluble ER proteins using ssHRP tagged with the ER retention motif (ssHRP ~EL) shows that it progresses through the Golgi stack no further than the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRP KDEL occurs via vesicular carriers as well as by tubular elements.
ssHRP has also been used to investigate the trans side of the Golgi complex, where incubation at reduced temperatures outlines the trans-Golgi network with HRP reaction product. Tracing the endosomal compartment with transferrin receptor in doublelabeling experiments with ssHRP fails to show any overlap between these two compartments. I N recent years the secretory pathway as originally outlined by Palade and his co-workers (reviewed by Palade, 1975) has been extensively elaborated upon. In particular, the Golgi complex is now thought to have well-developed tubular networks co-extensive with its cis- (Schweizer et al., 1988;Hsu et al., 1991) and trans- (Griffiths and Simons, 1986;Luzio et al., 1990;Geuze and Morr6, 1991) cisternae. The relationships between this organelle and the endoplasmic reticulum (as indicated by KDEL processing; Pelham, 1991) and the endocytic pathway (as indicated by the glycosylation of proteins recycling from the plasma membrane; Snider and Rogers, 1985;Green and Kelly, 1992) now need to be re-evaluated in detail (Mellman and Simons, 1992). In this re-evaluation the continuities induced by the fungal metabolite brefeldin A (Lippincott-Schwartz et al., 1989;Wood et al., 1991), the extensive interconnections being demonstrated in living cells by high-resolution microscopy (Cooper et al., 1990;Hopkins et al., 1991), and the domains defined by various cytoplasmic coatings (Weidman et al., 1993) will also need to be taken into account.
Previous morphological studies of the exocytic pathway have been constrained by the availability of methods sensitive enough to detect the low levels of endogenous protein existing within its various compartments. This is especially true for proteins in the earlier, pre-Golgi elements of the endoplasmic reticulum and transitional cisternae. For many studies this problem has been successfully circumvented by introducing exogenous protein into the secretory pathway by viral infection (Saraste and Kuismanen, 1984;Griffiths and Simons, 1986;Hedman et al., 1987). For the detailed analysis required for future work, however, probes which generate an amplified reporter signal and can be used at levels of expression which correspond more closely to endogenous trafficking proteins will be required.
Our approach to meeting these requirements has been to introduce chimeric proteins containing HRP into exocytic pathways by cDNA expression. The enzyme activity of this probe allows considerable signal amplification, an advantage widely exploited in its use as an exogenous, fluid phase marker in the endocytic pathway (Graham and Karnovsky, 1966;Tooze and Hollinshead, 1991) and which has also allowed it to be identified as an endogenous constituent within the exocytic pathway of cell types synthesizing peroxidase (Bainton and Farquhar, 1970;Herzog and Miller, 1972). An important advantage of the HRP reaction product, when generated in the soluble phase within intracellular compartments, is that it remains contained within the lumen of the cisterna or the vesicle in which the enzymes resides. In thick, unstained sections this usually allows even the most attenuated connections between compartments to be detected. A further, widely appreciated advantage of HRP as a morphological probe is its ability to remain enzymically active after being conjugated to a variety of protein ligands (Hopkins, 1985). The cDNA expression approach therefore also has the potential of being extended to chimeric proteins in which HRP can be incorporated within integral membrane proteins.
In this report we have evaluated the use of HRP as a probe for the exocytotic pathway, and have focused, in particular, on entry into the cis and exit from the trans cisternae of the Golgi complex. We show that HRP is active from the earliest part of the secretory pathway and that it can be efficiently secreted from cells, thus acting as an excellent tracer for the exocytic pathway. Using a form of HRP carrying the ERretention motif KDEL (Lys, Asp, Glu, Leu), we identify the cis-Golgi elements responsible for processing KDELbearing proteins, and identify vesicles and cisternal elements involved in both anterograde and retrograde traffic between the ER and the Golgi complex. Finally, we also show that the tubular elements of the trans-Golgi which are distributed throughout the pericentriolar area are separate from the closely adjacent tubules of the recycling endocytotic pathway.

Transfections
Cos'/and HEp2 cells were transiently transfected by electroporation (Chu et al., 1987) using an electroporator (Bio-Rad, Hertfordshire, UK). The day before transfection cells were seeded to "~75 % confluence with minimal cell-cell contact. On the day of transfection each 9-cm dish of cells was trypsinized and washed in DME containing 10% FCS, followed by HeBs (20 mM Hepes, pH 7.05, 137 mM NaC1, 5 mM KCI, 0.7 mM Na2HPO4, 6 mM D-glucose) and finally resuspended in 250 t~l HeBs. 10/~g of DNA (pSR~. ss.HRP or pSR~.ss.HRP KwL) was added and the DNA/cell mix was dectroporated (one pulse) in 0.4-cm cuvettes (Bio-Rad) at 300 V, 125 microfarads, and infinite ohms. The cells where then reseeded into the same size dishes and assayed for expression or processed for EM after 3 d in nonselective medium.
Stable lines of HEp2 and CaCo2 cells were produced by lipofection (Feigner et al., 1987;Chang and Brenner, 1988). The day before transfection cells were seeded to ,'v75% confluence with minimal cell-cell contact. On the day of transfection 10 t~g DNA (pSRt~.ss.HRP or pSRct. ss.HRP g°eL) was diluted to 250 #1 in OPTI-MEM and 100 #1 lipofectin was diluted to 250 td in OPTI-MEM (GIBCO BRL, Life Technols. Inc., Gaithersburg, MD). The DNA and lipofectin were mixed and the complex allowed to form at room temperature for 5 to 10 min. The mixture was then pipetted onto the ceils (pre-washed with OPTI-MEM to remove traces of serum), incubated at 37°C for 20 min followed by the addition of 2 ml serum-free DME, and the incubation then continued for another 5 h. DME containing FCS was then added and Ceils cultured for 1 (HEp2) or 3 d (CaCo2 cells), followed by trypsinization, and plating into six 96-well plates in the presence of selective agent (0.5 mg/ml G418; GIBCO BRL [Life Technologies Inc.] for HEp2 or 1 mg/ml for CaCo2 cells).

DNA Constructs
DNA manipulations were carded out by standard procedures, using reagents according to manufacturer's instructions.
Construction ofpSRa.ssHRP A construct encoding HRP isoenzyme c was artificially constructed by overlapping oligonucleotides (Ortlepp et al., 1989) on the basis of its published amino acid sequence (Welinder, 1979). This construct (pSA247) was kindly provided by Amersham Intl. (Amersham, UK). To enable entry into the secretory pathway, the signal sequence from human growth hormone (Hall et al., 1990) was added. The signal sequence (sshGH) was removed frQm the polylinker site of pMTL22p (Chambers et al., 1988) where it had been inserted as a BamI-II/HindlII fragment to produce pEKSP. This construct was kindly provided by H. J. Gilbert (University of Newcastle upon Tyne, Newcastle, UK). pEKSP was linearized with HindlII and the ends were blunted with the Klenow fragment of DNA polymerase. The DNA encoding HRP was excised from pSA247 by EcoRV/Hpal digestion and ligated into the blunt-ended HindlII site, downstream of sshGH to yield pEKSP.HRP. The fragment encoding an inframe fusion of the sshGH with the mature HRP was removed from pEKSP.HRP by EcoRV/SphI digestion. The 3' overhang of the SphI site was removed by T4 DNA polymerase and the resultant fragment ligated into the pSRc~ plasmid (Takebe et al., 1988; a kind gift from DNAX, Palo Alto, CA) which had been linearized by BamH1 and blunted with the Klenow fragment of DNA polymerase to give sshGH fused to the second amino acid (leucine) of mature HRP.
Construction of PSR~.ssHRP r°Et. HRp was generated from BBG10 (R and D Systems, Oxford, UK) by PCR amplification using the oligonucleotides: 5' G GAA TIC CAG TTA ACG CCG ACT Tq~ 3' and 5'C CCC AAG CTT AGA GTT GAC 3' such that the resulting fragment was flanked by EcoRI and HindlII sites.
The c-myc KDEL sequence was obtained from the sequence, 5'TTG GAG CAA AAG CTC ATT TCT GAA GAG GAC TTG AAG GAC GAA CTT TAA GCT 3' by PCR amplification using the oligonucleotides: 5' C CCC bAG CTT GAG CbA bAG CTC ATT TCT GAb, GAG GAC 3' and 5' C CCG CTC GAG TTA AAG TTC GTC CTT CAA 3' such that the resulting fragment was flanked by HindlII and XhoI sites.
The HRP and c-myc KDEL fragments bearing the relevant cohesive ends were ligated together and the product electrophoresed into LGT agarose (FMC Corp. BioProducts, Rockland, ME): The appropriate ligated product was then ligated into PSRc~.ss (produced by cloning the signal sequence from pEKSP as a StuI/EcoRV fragment, into pSRa at the XhoI site which had been blunt-ended using the Klenow fragment of DNA polymerase) which had been precleaved with EcoR/and Xhol and the ends dephosphorylated using calf intestinal alkaline phosphatase (Boehringer Mannheim Biochemicals, Indianapolis, IN).

HR P A ssay
To measure cell-associated HRP activity, transfected or control cells grown on 9-cm dishes were rinsed three times in PBS containing 0.5% BSA and suspended in 1 ml of 50 mM Tris/C1 (pH 2.5). Cells were then freeze/ thawed three times in a methanol/dry ice bath and centrifuged in a microfuge for 5 rain at 13,000 rpm. Media samples were clarified by centrifugation (3,000 rpm, 10 min) and then treated as for cell samples. Reactions were carried out in 50 mM Tris/Cl (pH 2.5) containing 0.1% o-phenylene diamine (Sigma, Poole, UK) and 0.02 % H202 (Sigma) at 370C in the dark. The reaction was stopped after 30 min by the addition of 1 M H2SO4 and the absorbance read at O1)450. As controls, buffer alone, mock-and HRPtransfected cells incubated in the absence of H202 were tested.

Electron Microscopy
Ceils were plated out on 3-cm petri dishes, cultured for 3 d in DME 10% FCS, and fixed directly or incubated for various times at 20°C or 15°C before fixation. Where reduced temperature incubations in a waterbath were carried out the medium was supplemented with 20 mM Hepes, pH 7.4. In some experiments cycloheximide (100 t~g/ml) or colchicine (2 #M) were added. In all experiments cells were rinsed with serum-free DME before fixation. In some cases, for the last hour at 20°C the medium was supplemented with anti-transferrin receptor antibody (B3czs) gold complexes prepared as described previously (Hopkins and Trowbridge, 1983). Fixation was carried out in Karnovsky fixative (2% paraformaldehyde, 2.5% glutaratdehyde in 100 mM sodium cacodylate, pH 7.5; Karnovsky, M. J. 1965. J. Cell Biol. 27:137a). They were incubated with H202 and diaminobenzidine prepared as described by Graham and Karnovsky (1966), rinsed, osmicated, dehydrated, and embedded by standard procedures (Hopkins and Trowbridge, 1983). Sections were cut at either 70 nm, stained with lead citrate, and uranyl acetate or cut 1-/~m thick, stabilized with a thin film of evaporated carbon, and viewed unstained in a Phillips CM12 transmission electron microscope. hGH, human growth hormone; c-myc, the epitope from c-myc recognized by antibody 9El0 that we used as a spacer; KDEL, the retention motif. Numbers of residues in elements shown as partial sequences are in brackets after the name. Note the single residue missing from the amino terminus of HRP in ssHRP.

HRP Can Be Efficiently Secreted from Animal Cells
We obtained a eDNA (pSA247) encoding mature HRP. To ensure entry of this enzyme into the secretory pathway we constructed a chimeric cDNA whereby the signal sequence from human growth hormone was attached to the amino terminus of HRP ( Fig. 1 and Materials and Methods). This chimeric cDNA, "ssHRP; was then cloned into the expression vector pSRo~ (Takebe et al., 1988) to create pSRa.ssHRP and transfected into Cos7 cells. Enzyme assays (not shown) indicated that a level of HRP activity 400-fold above background resulted from this transient expression.
We then attempted to establish stable lines of HEp2 cells and CaCo-2 cells expressing ssHRP. This was successful although few of the cell lines we established survived beyond 10 passages. The onset of mortality seemed to correlate with the level of peroxidase expression, implying that prolonged exposure to the enzyme could not, for some reason, be tolerated. Despite this problem, 10 passages provide sufficient cells for most studies and, as shown by electron microscopy, there is no evidence of degenerative change during these earlier passages.
To test if the ssHRP was entering the exocytic pathway, we investigated whether the HRP activity was efficiently secreted from Cos7 cells. Our experiments were designed to determine whether ssHRP was cleared from the cell by assaying for enzyme activity in the medium in the absence of continuing protein synthesis. Significant pools of activity remaining within the cell would suggest that the protein was being diverted and sequestered away from the constitutive secretory pathway and would complicate the interpretation of EM experiments.
Cos7 cells were transfected to produce transient expression of ssHRP. They were then rinsed and incubated with cycloheximide for varying lengths of time and at each point enzyme activity in the cells and medium was determined. As Fig. 2 shows, HRP activity is secreted from the cells with a tl/2 of '~80 rain, while intracellular activity is reduced to background levels within 4-5 h. All of the intracellular activity can eventually be recovered in the medium suggesting were transfected with pSR~/ssHRP and left for 36 h for plasmid replication to increase expression. Cells were then washed three times in DME/FCS supplemented with 0.2% BSA, and then incubated in the same medium for varying times in the presence of cycloheximide (100 izg/ml). HRP activity (mean + range, n = 3) in both medium (,,) and cells (t~) was assayed as described (Materials and Methods).
that intracellular degradation is negligible. Parallel experiments with HEp2 cells transfected with pSRa/ssHRP gave similar results (not shown).

Morphology of the Secretory Pathway between the RER and the Golgi Stack
Having shown that intracellular HRP activity is present within the secretory pathway of transfected cells, we carried out EM peroxidase cytochemistry. Cos7 cells expressing high levels of ssHRP show reaction product throughout the nuclear envelope and the cisternae of the RER. Golgi cisternae are also strongly stained but mitochondria, endocytotic elements and the plasma membrane are entirely negative (Fig. 3 a). In stably transfected CaCo-2 cells ssHRP reaction product is most strongly displayed in flattened Golgi cisternae ( Fig. 3 b). Where the section plane cuts across the flattened Golgi stack a gradient of reaction product, (concentration increasing towards the trans side) is often seen. Small (50-nm diarn) ssHRP-containing vesicles, which presumably represent constitutive secretory vesicles, are also found in the trans-Golgi area.
In HEp2 cells expressing ssHRP incubated at 37°C, HRP activity was barely detectable by electron microscopy. We therefore reduced the incubation temperature to 20°C, inducing an increase in the level of intracellular HRP. The increase in HRP activity which occurs in HEp2 cells incubated at 20°C arises because the release of activity is inhibited; on returning to 37°C in the presence of cycloheximide this HRP activity is secreted with a tla of 20 min and the enzyme is cleared from the cell within 120 min. Fig. 4 shows a Golgi area ofa HEp2 cell expressing ssHRP incubated for 4 h at 20°C in which HRP reaction product is detectable within the trans-most cisternae of the Golgi stack  Fig. 10) the distribution of HRP reaction product is discrete and clearly delineates the limiting membrane of the compartment which contains it. It is also worth noting that the membranes themselves and any associated cytoplasmic coatings are clearly displayed.
We believe the increase in reaction product toward the trans side of the Golgi stack arises because 20°C blocks exit from the TGN (Gritfiths and Simons, 1986) but that the process(es) which concentrate secretory product continue to operate within compartments proximal to this block. Because cells are fixed and washed before the DAB reaction is carried out, and local gradients of small molecules and ions will be dissipated during this processing, the intensity of the staining resulting from HRP activity is not affected by local physiological conditions (such as pH). The staining intensity of lumenal content should, therefore, directly reflect the amount of enzyme present within the compartment in which it is contained.
To examine the transfer of soluble phase tracer between the RER and the Golgi stack in more detail we constructed an HRP chimera which carried a KDEL retention signal at the COOH terminus; "ssHRP ~L " (Fig. 1). The extent to which ssHRP and ssHRP K°EL are retained within HEp2 cells during a 24-h incubation was compared (Fig. 5). It is evident, as expected (Munro and Pelham, 1987), that the presence of the KDEL tetrapeptide causes an intracellular accumulation of the tracer. However, a significant amount of ssHRP KDEL was released into the medium in some of these experiments, suggesting that the capacity of the KDEL retention system can be overloaded in HEp2 cells at relatively modest levels of expression.
EM cytochemistry of HEp2 cells transfected with pSRa. ssHRP KDEL and incubated at 37°C showed HRP reaction product distributed evenly throughout the endoplasmic reticulum and nuclear envelope. In addition to RER cisternae the cis-most cisternae of the Golgi stacks also contained DAB reaction product (Fig. 6). Within the cis-most cisternae it is sometimes restricted to short stretches of the flattened cisternae or to their bulbous termini (Fig. 7, a and b). In thin sections there are also frequent profiles to suggest continuity between the Golgi stack and adjacent cisternae which may belong to the ER (Fig. 7 a).
In addition to the flattened cisternae of the RER there were also HRP-positive 50-nm-diam vesicles which often seemed to be in continuity with the RER or the nuclear envelope (Fig. 7 d). Sometimes these vesicular elements appeared to be connected to one another by narrow tubules so that they resembled small bunches of grapes. Thin sections suggested that a proportion of these structures are free vesicles (Fig.  7 c) and thick sections up to 10 times their diameter (not shown) showed that some were, indeed, unconnected to their neighbors. The diaminobenzidine (DAB) t reaction product within the 50-nM-diam vesicles filled the lumen and was often considerably more electron opaque than the DAB reaction product distributed in the adjacent RER cisternae.
The only other class of DAB-positive structures in cells transfected with ssHRP KDEL were 80-100-nm-diam vesicles (Fig. 6). These larger vesicles did not have as consistent a size or as distinctively spherical a shape as the 50-nm vesicles, often they were ovoid in form. They were grouped in clusters and scattered amongst the cisternae of the ER throughout the cytoplasm as well as in the vicinity of the cis-Golgi complex. Within the clusters adjacent to the RER some profiles showed direct continuity between the 80-nm vesicles containing dense DAB reaction product and ER cisternae (Fig.  6 c). In the Golgi area these vesicles were sometimes single and clearly adjacent to the cis-Golgi complex (Fig. 7 a), on other occasions they had an elongated, sinuous profile and were indistinguishable from cis-most Golgi cisternal elements (Fig. 6, a and b).
The intensities of DAB staining in these various elements are characteristic. In the RER and 50-nm vesicles and associated tubules, a moderate level of staining is usually seen.  . EM of HEp2 cell expressing ssHRP at reduced temperature. HEp2 cells stably expressing ssHRP were incubated at 20°C for 4 h and then processed for EM peroxidase cytochemistry. This section shows DAB reaction product distributed as a discrete precipitate of moderate electron opacity on the lumenal side of the trans Golgi eisternal membranes (arrowheads). The cis-most Golgi cisternae (large arrows) are negative as are most ER cisternae. Small arrows indicate DAB staining of an ER transitional dement and an adjacent 50 nm vesicle. A coated bud (cb) on a trans-Golgi cisterna is indicated. Bar, 0.2 t~m.
In the cis-Golgi and the 80-nm vesicles a noticeably higher level of staining is the norm, although this staining is less evenly distributed, often concentrated at the perimeter membrane.
Previous work on the processing of KDEL-bearing cathepsin (Pelham, 1988) has shown that temperatures in the region of 14.5-15 °C inhibit the processing of its oligosaccharides characteristic of delivery to the Golgi stack. When HEp2 cells transfected with ssHRP KD~L are incubated at 15°C the distribution of the tracer is altered (Fig. 7 e). Within 2 h reaction product becomes barely detectable in the RER, compact clouds of 50-nm-diam vesicles scattered throughout the cytoplasm appear, and the 80-100-nm-diam vesicles which are such a prominent feature at 37°C are much reduced in number. Within the groups of 50-nm vesicles only a minority stain with DAB (Fig. 7 e).
With longer incubations at 15°C the form of the Golgi complex changes dramatically; the flattened, stacked cisternae curl to form compact, concentric whorls and the cytoplasm in the vicinity of these cisternal whorls becomes filled with vesicles of various sizes (Fig. 8). Thin section profiles suggest that the vesicles are derived from the breakdown of the Golgi stack during the 15°C incubation. It is of interest, therefore, that within these clouds of vesicles the ssHRP K°EL vesicles remain separate and identifiable even after an incubation of 20 h at 15°C (Fig. 8).

The Morphology of the Secretory Pathway beyond the Golgi Stack
At 20°C ssHRP accumulates in the Golgi stack. In addition to the flattened stacks, HRP reaction product is also distributed in a variety of vacuoles, branching 60-nm-diam tubules and flattened saccules in the Golgi area. These HRPpositive tubules lie on the concave side of the stack and presumably represent elements of the trans Golgi reticulum. By cutting thick (1 /~m) sections and viewing them unstained (not shown) it is clear that many of these HRP-positive elements are part of an extensive tubular reticulum distributed throughout the pericentriolar area.
In HEp2 cells there is a well-developed endocytotic compartrnent in the pericentriolar area which also consists of branching, 60-nm-diam tubules (Hopkins and Trowbridge, 1983;Hopkins, 1985). For further detailed analysis of this part of the pathway it was, therefore, of interest to determine the spatial relationship between these two tubular systems. This was examined by incubating HEp2 ceils expressing ssHRP at 20°C for 60 min with anti-transferrin receptor antibody (B3as)-gold complexes. As has been shown in previous studies (Hopkins and Trowbridge, 1983;Hopkins et al., 1991) this gold tracer outlines the endocytotic pathway and, in particular, identifies the tubules of the recycling pathway located in the pericentriolar area (Hopkins et al., 1994). Electron microscope cytochemistry for HRP shows both the ssHRP and the gold tracers distributed in tubular/vesicular elements in the trans-Golgilpericentriotar area. While the labeled vesicles often lie very close to each other and thick sections suggest that the labeled tubules are frequently intertwined, neither double-labeled tubules nor vesicles were observed (Fig. 10 a). However, at 20°C, transport of ssHRP beyond the trans-Golgi towards the cell surface is prevented and in subsequent experiments the 20°C incubation was therefore followed by a chase at 37°C (in the continuing presence of B3as gold, but with further HRP synthesis prevented by the addition of cycloheximide). With the transfer to 37°C, HRP activity rapidly drained from the Golgi complex whilst the pericentriolar elements of the endocytotic pathway remained extensively labeled with gold. Under these conditions double-labeled elements were still not observed (Fig. t0, b and c). We conclude, therefore, that the trans-Golgi reticulum and the tubules of the endocytic pathway in the pericentriolar area are separate systems. The transfer between endosomes and the trans-Golgi which has been reported previously (e.g., Snider and Rogers, 1985;Geen and Kelly, 1992) probably depends upon a selective, receptor-mediated, concentrative step and this might well be difficult to detect with a fluid phase tracer like ssHRP.

Discussion
In this paper we show that introducing HRP into the RER allows constitutive pathways into and out of the Golgi stack to be examined in detail. The newly synthesized HRP is enzymically active from the time it is translocated into the lumen of the RER and it is potentially possible, therefore, to trace the entire exocytotic pathway. The kinetics with which ssHRP is cleared from the HEp2 and cos7 cells are similar to those observed for other proteins traveling in the soluble phase (e.g., Lodish et al., 1983). This, together with EM cytochemistry which shows no indication of HRP in pathways other than the exocytotic route (lysosomes are, for example, negative at all times) clearly suggests that the ssHRP is a reliable fluid phase marker for the exocytic pathway.
In previous electron microscopical studies of KDELbearing proteins a variety of localizations have been obtained. In plants (Napier et al., 1992), and Purkinje cells (Yamamoto et al., 1991) immunogold electron microscopy identified KDEL proteins only in the ER, whereas in the exocrine pancreas (Takemoto et al., 1992) these proteins were found distributed throughout the entire secretory pathway. Using ssHRP t°~L, we find reaction product within the ER, transport vesicles, and the cis-Golgi at 37°C. Our data thus conform with biochemical observations which show that KDEL-bearing proteins are transported from the RER to the Golgi complex before retrieval (Pelham, 1988), and with the distribution of erd2 as seen by immunofluorescence  and EM (Tang et al., 1993).
Our data emphasize the extent to which KDEL-tagged proteins can traffic through a variety of different membranebound compartments. Moreover, the staining levels we ob-  The Journal of Cell Biology, Volume 127, 1994 Figure 9. The effect of colchicine on a HEp2 cell expressing ssHRP KDEL. HEp2 cells stably expressing ssHRP goEL were treated with colchicine at 2/zM for 1 h before fixation and processing for EM. (a) Low power view shows that DAB reaction product is distributed throughout the ER and within 80-100-nm-diam vesicles which are grouped in clusters throughout the cytoplasm (arrows).
Note that this cell has a morphology typical of mitotic cells which have not been colchicine treated. (b) Enlargement of area shown in a revealing details of accumulated DAB-positive vesicles. Bars, 2.0/zm. serve may suggest which of these structures lie before and which lie after interaction of the KDEL-ligand with its receptor. If, as suggested, the binding ofligand to erd2 is associated with an oligomerization into patches of the receptor Townsley et al., 1993), thereby enhancing the concentrative effect of a soluble ligand binding to a membrane-bonnd receptor, then the higher levels of staining observed with ssHRP K°~L indicate which elements contain receptor-bound tracer. It should be noted that the regions of high concentration we see within the cis-Golgi cisterna, (especially in buds) are similar in size and enhanced staining to the 80-nm vesicles distributed around the Golgi complex, scattered throughout the cytoplasm and in continuity with ER cisternae. Together these elements probably outline the retrograde pathway followed by KDELligand-erd 2 complexes from the cis-Golgi to the ER.
The 50-nm-diam vesicles which also stain strongly with DAB could also be on the KDEL retrograde pathway. However, the tubular connections which are often seen to attach them to the RER and nuclear envelope suggest that they are in the process of forming rather than fusing with these cisternal elements. Moreover, the homogeneous distribution of reaction product within these vesicles is very different from the membrane associated staining seen in the 80-nm-diam elements. The increased DAB staining observed in 50-nm vesicles may not, therefore, be due to the HRP ligand bound to the KDEL receptor but may, instead, indicate a less specific, concentrative step taking place on the anterograde pathway. The existence of a concentrative vesicular step on the anterograde pathway between the ER and the cis-Golgi has recently been proposed for both a soluble, secretory protein, and a trafficking viral membrane protein (Balch et al., 1994;Mizuno and Singer, 1993).
One of the effects of colchicine that we observed was an increase in the number of 80-nm-diam vesicles. This would be consistent with the view that these vesicles are on the retrograde pathway, since it has been shown that other microtubule-disrupting drugs such as nocodazole and griseofulvin also block transport in this direction (Lippincott-Schwartz et al., 1990). However, the colchicine treatment also leads to an accumulation of ssHRP KDEL in the RER suggesting, in agreement with the work of Saraste and Svensson (1991), that microtubule depolymerization also has an influence on transport in the anterograde direction.
The relationship between the compartments containing ssHRP sDEL which we have identified, and the elements identified in previous studies of transport between the RER and the Golgi complex will need to be addressed in future work. At the present time it is not possible to obtain a coherent view of how the intermediate compartments identified by marker proteins such as p53 and p63 (Schweitzer et al., 1988(Schweitzer et al., , I990, 1993 relate, for example, to the structures involved in the transfer of viral membrane proteins out of the RER (Hobman et al., 1992). From the effects observed at lowered temperatures and following treatment with microtubule-depolymerizing agents (Saraste and Knismanen, 1984; Figure 8. HEp2 cells expressing ssI-IRP KDEL during incubation at 15°C. HEp2 cells stably expressing ssHRP KDEL were incubated for 20 h at 15°C before fixation and processing for EM peroxidase cytochemistry. The section shows Golgi complexes consisting of whorls of cisternae and a mitochondrial morphology characteristic of the effects of lowered temperature on these cells. Several kinds of vesicles are evident: the small arrowheads indicate 50-nm-diam vesicles lacking DAB, the larger arrowheads indicate coat material which is probably free in the cytoplasm, the arrows indicate ,'~80-nm-diam vesicles containing DAB reaction product associated with the inner face of their perimeter membranes. In thin sections like this it is difficult to determine if the smaller positively stained profiles are 50-nm-diam vesicles or tangential sections cutting across the periphery of*80-nm-diam vesicles. RER cisternae are no longer stained with DAB at this lowered temperature. Bar, 2.0/~m. Figure 10. HEp2 cells expressing ssHRP that have been allowed to endocytose colloidal gold coupled to antibodies that recognize the transfen'in receptor. (a) HEp2 cells stably expressing ssHRP were incubated for 2 h at 20°C in the presence of Bv25 gold complexes before fixation and processing for EM. This section shows that HRP reaction product is distributed throughout the Golgi stack and within elements in the trans-Golgi area. Gold conjugates (arrows) clearly identify the tubules and vacuoles of the endocytic pathway. Lysosomes are not labeled by either tracer. (b) As in a but cells were warmed to 37°C for 10 min in the continued presence of gold conjugate before fixation. Gold-loaded elements are not labeled by ssHRP. (c) As in a but cells were warmed to 37°C in the continued presence of gold conjugate for 30 min before fixation. There are numerous gold-labeled endocytic elements (arrows) surrounding the centriole (C). The trans-Golgi elements are largely free of DAB reaction product but some (/arge arrows) can be identified. Lysosomes (L) remain unlabeled and there are no elements labeled with both gold and HRP. Bars, 0.2 t~m.
The Journal of Cell Biology, Volume 127, 1994 Saraste and Svensson, 1991;Plutner et al., 1992;Schweizer et al., 1993;Krijnse-Locker et al., 1994;Balch et al., 1994) it is probable that many of the structures described by others as belonging to the intermediate compartment are the same as those described here. However, it should be emphasized that while previous high-resolution studies have described only anterograde traffic between the RER and the Golgi complex, our ssHRP zDEL tracer outlines compartments involved in both anterograde and retrograde traffic.
Further progress will probably require comparative analyses in which soluble phase proteins (with and without RER retention signals) and membrane proteins are localized simultaneously.
The staining of ssHRP across the Golgi stack suggests an increasing concentration of this tracer in the more trans elements. A concentration of a fluid-phase marker as it traverses the Golgi stack has been reported before (e.g., Bendayan et al., 1980;Pelham, 1989). This increase in concentration would arise if retrograde pathways have a relatively low fluid-phase capacity, but there is as yet no indication that retrograde-directed vesicles are of lower carrying capacity (e.g., smaller) than anterograde vesicles. Alternatively, the increased staining may be due to some other process such as the removal of water from this part of the anterograde pathway.
The distribution of ssHRP reaction product at 20°C clearly identifies the form and extent of the trans-Golgi compartment in HEp2 cells. This compartment, originally defined as the TGN in studies using viral proteins (Griffiths and Simons, 1986), is found in the pericentriolar area. Biochemical analyses have revealed in more than one cell type and with several different surface proteins (e.g., Snider and Rogers, 1985;Green and Kelly, 1992) that there is trafficking between the endocytic pathway and the TGN. Experiments with brefeldin A (Wood et al., 1991; Lippincott-Schwartz et al., 1991; Reaves and Banting, 1992) also suggest a close relationship between these two compartments. However, there is little evidence from previous morphological observations that direct continuity exists between them. Our analysis confirms the work of Geuze and others (Geuze and Morrd, 1991) that an extensive tubular reticulum exists in the trans-Golgi area at 20°C in the absence of viral infection. However, our HRP reporter technique, which should allow detection of even the most narrowly attenuated continuities between cisternae, also fails to identify connections between this compartment and the endosome.
In addition to defining the constitutive exocytic route HRP reporters clearly have wide potential for other pathways. By coupling targeting signals from other proteins to HRP, most of the intracellular pathways which cannot be accessed from the cell surface can be examined. By coupling membrane anchors and transmembrane segments carrying cytoplasmic domains of membrane proteins containing targeting signals to HRP membrane proteins trafficking throughout the cell can be studied. We have already shown that eDNA chimeras between HRP and transferrin receptor and sialyl transferase segments are enzymatically active (unpublished observations): their trafficking is currently being investigated.
Finally, in addition to these morphological studies, it should be possible to exploit the ability of HRP to render colocalized proteins insoluble as in previous studies of the endocytic pathway (Courtoy et al., 1984;Ajioka and Kaplan, 1987;Stoorvogel et al., 1988;Stoorvogel et al., 1989;Stoorvogel et al., 1991). This would be extremely useful for quantitative studies of protein sorting between exocytic routes.


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Members of the fatty acid binding protein family are differentiation factors for the mammary gland.

Mammary gland development is controlled by systemic hormones and by growth factors that might complement or mediate hormonal action. Peptides that locally signal growth cessation and stimulate differentiation of the developing epithelium have not been described. Here, we report that recombinant and wild-type forms of mammary-derived growth inhibitor (MDGI) and heart-fatty acid binding protein (FABP), which belong to the FABP family, specifically inhibit growth of normal mouse mammary epithelial cells (MEC), while growth of stromal cells is not suppressed. In mammary gland organ culture, inhibition of ductal growth is associated with the appearance of bulbous alveolar end buds and formation of fully developed lobuloalveolar structures. In parallel, MDGI stimulates its own expression and promotes milk protein synthesis. Selective inhibition of endogenous MDGI expression in MEC by antisense phosphorothioate oligonucleotides suppresses appearance of alveolar end buds and lowers the beta-casein level in organ cultures. Furthermore, MDGI suppresses the mitogenic effects of epidermal growth factor, and epidermal growth factor antagonizes the activities of MDGI. Finally, the regulatory properties of MDGI can be fully mimicked by an 11-amino acid sequence, represented in the COOH terminus of MDGI and a subfamily of structurally related FABPs. This peptide does not bind fatty acids. To our knowledge, this is the first report about a growth inhibitor promoting mammary gland differentiation.

Abstract. Mammary gland development is controlled by systemic hormones and by growth factors that might complement or mediate hormonal action. Peptides that locally signal growth cessation and stimulate differentiation of the developing epithelium have not been described. Here, we report that recombinant and wild-type forms of mammary-derived growth inhibitor (MDGI) and heart-fatty acid binding protein (FABP), which belong to the FABP family, specifically inhibit growth of normal mouse mammary epithelial cells (MEC), while growth of stromal cells is not suppressed. In mammary gland organ culture, inhibition of ductal growth is associated with the appearance of bulbous alveolar end buds and formation of fully developed lobuloalveolar structures. In parallel, MDGI stimulates its own expression and promotes milk protein synthesis. Selective inhibition of endogenous MDGI expression in MEC by antisense phosphorothioate oligonucleotides suppresses appearance of alveolar end buds and lowers the ~-casein level in organ cultures. Furthermore, MDGI suppresses the mitogenic effects of epidermal growth factor, and epidermal growth factor antagonizes the activities of MDGI. Finally, the regulatory properties of MDGI can be fully mimicked by an l 1-amino acid sequence, represented in the COOH terminus of MDGI and a subfamily of structurally related FABPs. This peptide does not bind fatty acids. To our knowledge, this is the first report about a growth inhibitor promoting mammary gland differentiation.
basement membrane (39,63), it has been documented that several steroid hormones in conjunction with prolactin and insulin regulate this process (reviewed in reference 66). Although the systemic importance of hormones such as estrogen, progesterone, prolactin, cortisol, and growth hormone in the development of the mammary gland has been well documented, these hormones virtually lack growth stimulatory or inhibitory activities in vitro (32,46). This has prompted a search for polypeptide growth factors that might locally mediate or complement action of systemic hormones. A number of growth factors that either stimulate or inhibit epithelial DNA synthesis in the murine mammary gland have been described (32). For example, mitogenic effects on MEC have been described for epidermal growth factor (EGF) (16,27,65,70) and TGF ot (55,59), demonstrating the importance of these growth factors for ductal growth and lobuloalveolar development. TGF-/~I has been shown to inhibit ductal outgrowth in the mammary gland of virgin mice (47,53). In addition, the same molecule can inhibit functional differentiation in vitro (41,50), and it impairs differentiation and milk protein synthesis in mice (34).
In contrast to these reports, there are no available data that describe growth inhibitory and differentiation stimulatory growth factor activities during functional differentiation of the mammary gland. When we screened for epithelial growth inhibitors, we have detected and purified from lactating bovine mammary gland (7) and from milk fat globule membranes (12) the mammary-derived growth inhibitor (MDGI) and a COOHterminally truncated form of MDGI (13). The 14.5-kD protein was sequenced (7), and its eDNA was cloned (38). Originally, MDGI has been discovered according to its growth inhibitory activities on breast cancer cells in vitro (8). Our studies on MDGI mRNA and protein expression in the developing bovine and mouse mammary gland revealed a strong correlation between expression of MDGI in MEC and differentiation (6,38,43). MDGI expression was found to be hormonally regulated (6), suggesting a local role of the growth inhibitor in mediating or complementing hormonal action during differentiation (23,24).
MDGI belongs to a multigene family of conservative proteins, referred to as fatty acid binding proteins (FABP), known to bind long-chain fatty acids, retinoids, and eicosanoids (reviewed in references 21, 40, 68, 69). A striking homology was evident between MDGI and the heart fatty acid binding protein (FABP) (H-FABP), which differ only in 10 positions of the amino acid sequence (7), and most likely represent isoforms that are encoded by the same gene (67). Other closely related members include the FABPs found in the brain and the cellular retinoic acid binding protein (CRABP) (52, 14, 56, 68). The function of these proteins is not yet defined, although a role in fatty acid transport, sequestration, or metabolism has been widely discussed (21,69). Evidence for the existence of physiologically interesting FABP ligands such as selenium (4), E-type prostaglandins (18), or hydroxyeicosatetraenoic acid (48), as well as mitosisassociated expression of liver FABP (36), gave rise to speculations of some role of FABP in growth control.
To address directly the question of a role for MDGI and H-FABP as growth inhibitors and differentiation factors of MEC, we have cultured primary mouse mammary epithelial cells and whole mouse mammary glands in presence of wildtype, recombinant as well as mutated MDGI and H-FABP forms. We show that MDGI and H-FABP inhibit epithelial DNA synthesis, promote morphological differentiation, and stimulate milk protein synthesis. Selective inhibition of MDGI expression in organ cultures suppresses alveolar budding and impairs r-casein synthesis. The MDGI and H-FABP properties do not require fatty acid binding, and can be fully mimicked by a peptide that comprises COOHterminal amino acids conservatively maintained in a subgroup of MDGI-related FABPs. This first report about a manunary epithelial differentiation factor suggests that other members of the FABP family might similarly to MDGI regulate growth and differentiation.

Cell and Organ Culture
Primary Cultures from Mammary Glands of Adult Virgin Mice. Mammary cells were collected after collagenase digestion of mammary glands excised from 8-to 13-wk-old mice. Approximately 105 cells were plated onto plastic culture dishes (60 mm in diameter) and maintained in DME supplemented with 10 % fetal calf serum, penicillin, streptomycin, 4 #g/ml of insulin, and 10 ng/rrd EGE Mammary Gland Organ Culture. Whole second thoracic mammary glands of hormonally primed virgin mice were cultured in serum-free medium according to Banarjee et al. (2). 4-wk-old BALB/c mice were injected subcutaneously for nine consecutive days with I nag progesterone and 1 #g estradiol (Jenapharm, Jena, Germany). The second pair of thoracic glands was excised and cultivated for 5 d as whole organ on perforated cyciopore membranes (No. 3090; Falcon Plastics, Cockeysville, MD) inserted into wells of culture plates (No. 3046; Falcon Plastics) in 2 ml medium 199 with Hanks' salts (Gibco BRL, Gaithersburg, MD) and 50 #g/ml gentamycin in an atmosphere of 50% 02/5% COz, with medium changes every 2 d. The medium was supplemented with aldosterone (No. 12050; Serva, Sigma, Germany), ovine-prolactin (31 IU/mg, No. L-6520; Germany), insulin (Berlin-Chemie, Berlin, Germany) (each at 5 #g/mi), and 1 #g/ml hydrocortisone (No. 24608; Merck, Darmstadt, Germany), and will be referred to in this paper as the APIH medium. This medium has been used in all experiments described below. EGF was prepared as described before (58). Generally, three pairs of contralateral glands representing control and test groups were examined.

Histology and Morphometry
Whole-mount hematoxylin stains of mammary glands were obtained as described (49). For histology, formalin-fixed glands were embedded in paraffin. 5-#m sections were cut, deparaffinized, stained with hematoxylin and eosin, and analyzed as described before (6). Histologic criteria used were as follows (6): for ductules, a linear coat of cubic epithelial cells without adjacent mesenchyme and signs of secretion; for alveoli, presence of secretory cells with vacuoles and luminal secretion. Only structures exhibiting lumina were counted. At least three pairs of contraiateral glands were examined. Fat droplet index was defined as the quotient of the number of alveoli consisting of >50% of epithelial cells with intraepithelini fat droplets to the total number of alveoli. Alveolar secretion was defined as the quotient of the number of aiveoli with lumina containing secretory products to the total number of alveoli. An experimental group comprised of ,~500 different structures for the morphometric analysis.

DNA Synthesis and Immunocytochemistry
Primary Cultures. To estimate DNA synthesis, cells were seeded at a density of 2 x 106 cells in 60-mm dishes in DME containing 10% FBS. 12 h later, the peptide to be tested was added. Cultures were maintained for 48 h before 5-bromo-2'-deoxyuridine (BrdU) at 10 #M was added according to the manufacturer's instructions (Boehringer Mannheim GmbH, Mannheim, Germany). For BrdU staining, cultures were washed three times with PBS and fixed in 70% ethanoi/glycine (50 mM, pH 2.0) for 30 rain at -20°C. Cells were first incubated with 1:10 diluted anti BrdU antiserum, followed by an incubation with a secondary anti-mouse IgG conjugated to alkaline phosphatase. Bromo chloro indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) was used as substrate. Colonies >1,000 cells were counted. On the average, 10-20 colonies were analyzed for BrdU immunostaining. Results (expressed as percent of the control, which received the appropriate buffer) were obtained from triplicate experiments (SD = +20%).
Mammary Gland Organ Culture. Growth inhibition was determined by labeling cells with BrdU (100 #M) for 4 h before cultures were terminated. Then organ cultures were fixed in 4% formalin/0.5% trichloroacetic acid before embedding. Deparaffinized sections were washed in PBS and analyzed as described above with the anti-BrdU antiserum kit (Boehringer Mannheim GmbH). Sections were counterstained with fast nuclear red and the labeling index (LI) estimated as described before (6). At least 1,000 nuclei were counted for each experimental group and histologic structure. Percent inhibition = 100-LI treated/LI controls x 100. Immunohistochemical analysis for expression of MDGI and t-casein in tissue sections of a mammary gland was performed as described before (38,43). Deparaffinized sections were analyzed with a rabbit anti-mouse t-casein antiserum or an afffinity-purified IgG fraction of a rabbit antiserum against bovine MDGI (43). For staining, sections were washed with PBS and treated with the streptavidin-biotinylated peroxidase complex (1:200) (Amersham International, Amersham, IL) using diaminobenzidine as substrate. Sections were counterstained with hematoxylin. glands were used exactly as described before (6). Total RNA was extracted, denatured, and applied to 1% agarose gels for transfer to Hybond C-extra membranes (Amersham International) by capillary blotting. Levels for mRNAs were estimated from Northern blots hybridized with 32p-labeled eDNA probes for MDGI,/~-casein, and whey acidic protein (WAP), and were normalized to 28S ribosomal RNA, as described earlier (6). The 0.4-kD eDNA probe in pBR327 for WAP was provided by Dr. N. Hynes (Friedrich Miescher Institute, Basel, Switzerland).

Synthesis and Application of Oligonucleotides
The 17-mer phosphothioate oligonueleotides were synthesized and purified Cells derived from mammary glands of adult mice were seeded at a density of 2 x 106 cells. 12 h later, wild-type MDGI was added at 10 -9 M. Cultures were maintained 48 h before BrdU was added for immunostaining (B). Effects of recombinant and wild-type FABPs on epithelial DNA synthesis. DNA synthesis expressed as the percent of inhibition was estimated from primary cultures treated with the corresponding MDGI and FABP forms and subsequently stained for BrdU incorporation as described before. None of the peptides inhibited stromal DNA synthesis. Native MDGI, recombinant MDGI, and liver FABP slightly stimulated DNA synthesis in stromal cells by 12, 18, and 19%, respectively. Native and recombinant FABP were inactive. Colonies >1,000 cells were counted. Proteins were added at 10-9 M. Results were obtained from triplicate experiments (SD = +20%). Bar, 50 #m. Immunodetection was performed with a rabbit anti-mouse/3-casein or anti-MDGI antibody visualized with a biotin/streptavidin detection system using diaminobenzidine as peroxidase substrate. Note marked/5-casein expression in ductal and alveolar MEC, and strong luminal/3-casein secretion if compared to MDGI. Bar, 100/~m. by high-performance liquid chromatography (BioTez Berlin Buch GmbH). Sequences correspond to the mouse MDGI cDNA (6) with the translation start as first codon of the sense or the complementary antisense strand. The base composition of the random oligonucleotide was made identically to the antisense probe. The modified oligonucleotides were directly added to the medium on day 1 of the culture. Cultures received the oligonucleotides with every medium change.

Synthesis of Recombinant Proteins
Including microheterogeneities found upon protein sequencing in five positions (7), MDGI differs from the H-FABP sequence deduced from cDNA cloning or protein sequencing (5,7,38)  Double-stranded M13mp18DNA was cut by Kpnl and HindIII. The vector pET-BHFABE which is a construct of the pET-3d (61) and ofa Ncol/HindIH fragment of the H-FABP-cDNA, was used for cloning the MDGI cDNA into an expression vector. To this end, an NcoI/HincRII fragment of the unmutated H-FABP cDNA (derived from pET-BHFABP) was replaced by the respective MDGI fragment. The resulting expression vector was named pET-BHMDGI. To express proteins, the pRT-3d vector in BL21 (DE3) cells was used (61). Soluble recombinant proteins were harvested from supernatants and isolated by CM Sephadex C50 and Sephacryl S-100 HR, as described (5). All amino acid substitutions were confirmed by protein sequencing as described (5,7). Recombinant forms for bovine liver FABP (L-FABP) and rat intestine FABP (I-FABP) were produced according to their sequences (68). The mutant human H-FABP carrying 106 Thr for Arg was prepared by site-directed mutagenesis using the uracil selection method (37).

Fatty Acid Binding
Fatty acid binding was measured at 2 #M oleic acid and 10/zg protein according to the lipidex method (22) as described by us earlier (9).

Peptide Synthesis
Synthesis of peptides with a peptide synthesizer (430A; Applied Biosystems, Inc., Foster City, CA), purification, and identification of purified peptides, has been described elsewhere (72).

MDGI and Heart FABP Specifically Inhibit Epithelial DNA Synthesis
Protein sequencing showed coexistence in the bovine mammary gland of H-FABP and of MDGI, which differ from each other in 10 amino acids (7). To test whether MDGI and H-FABP both possess growth inhibitory activity and whether growth inhibition is specific for epithelial cells, the respective recombinant and wild-type proteins were produced, and were compared in activity in primary cultures of MEC (Figs. 1, A and B). As shown, wild-type MDGI at 10 -9 M specifically inhibited DNA synthesis in MEC ( Fig.  1 A c) if compared to the control ( Fig. 1 A a), whereas growth of mammary stromal cells was unaffected ( Fig. 1 A,  b and d). Wild-type and recombinant MDGI-or H-FABP inhibited DNA synthesis of MEC by ~50-60% (Fig. 1 B). Half-maximal inhibition was obtained with ~10 -1° M of the MDGI or H-FABP forms (not shown). Recombinant L-and I-FABP, which represent more distant members of the FABP family and share an overall sequence homology of 44-55 % with MDG! (7), had no significant growth inhibitory effect (Fig. 1 B). Therefore, growth inhibition seems to be specific for MEC and for MDGI and the heart type of FABP.

MDGI Inhibits Ductal Growth and Promotes Functional Differentiation
To ascertain whether MDGI might regulate differentiation, whole-organ mammary glands that were derived from virgin mice were cultured for 5 d in a serum-free medium containing aldosterone, prolactin, insulin, and hydrocortisone (APIH medium) (Fig. 2). This hormone milieu results in complete lobuloalveolar development of the gland and terminates in functional differentiation. Under the same conditions, organ cultures were treated for 5 d with MDGI at 10 -9 M. The presence of MDGI resulted in appearance of smaller ducts and ductules with numerous side branches (Fig. 2 B) when compared with the control (Fig. 2 A). H-FABP produced essentially the same morphological changes (not shown). Accordingly, when labeling indices were estimated in alveoli, ducts and ductules of MDGI-or H-FABP-treated mammary glands, strongest inhibition of DNA synthesis (*60% if compared to the untreated gland) was found in ductular MEC (Table I). In treated cultures, ductules were terminated by bulbous alveolar end buds (Fig.  2 D) when compared with the control gland (Fig. 2 C). The alveolar end buds represent a developmental pathway that eventually leads to secretory alveoli at functional differentiation. Indeed, histological examination of MDGI-treated glands revealed the appearance of mono-layered secretory active alveoli with enlarged luminal spaces (Fig. 2, E and F).
In accord with these changes, which are characteristic for the differentiated phenotype, functional differentiation evidenced by B-casein expression was stimulated (Fig. 2, G and H). Intracellular and luminal B-casein levels, which can be considered to indicate an earlier period of functional differentiation, were elevated (Fig. 2 H). MDGI treatment produced an autostimulation of its mRNA expression (Table  II). Correspondingly, a clear increase in endogenous MDGI production was detected in MEC and, to a much less extent, in the luminal spaces (Figs. 2, I and J). The striking differences between the pattern of the B-casein and MDGI expression exactly reflect the situation we have described for the developing mammary gland (6). Substantiation that MDGI, as well as the human and bovine forms of H-FABP, might function as differentiation factors in the mammary gland was assessed by quantifying "early" and "late" markers of functional differentiation such as B-casein and WAP expression, fat droplet accumulation, and secretory activity (Table ID. As shown, MDGI and H-FABP elevated the levels of markers for functional differentiation up to threefold.

Inhibition of MDGI Expression Suppresses Alveolar Differentiation and f3-Casein Expression
In mammary glands of primed virgin mice, the MDGI gene is not expressed; however, if these glands are taken in culture in presence of APIH, MDGI expression can be first detected after 3 d (6). Hormonally induced MDGI expression, which reaches some maximum after 8 d (6), follows closely the pattern that has been found during pregnancy (6,38). This finding argues in favour of a local role for endogenous MDGI during growth and differentiation, and we have therefore tested whether MDGI expression is a prerequisite for normal development. To this end, antisense phosphothioate oligonucleotides complementary to position 1-17 (relative to the translation initiation side) of the murine MDGI coding region (6) (as-oligonucleotide) were added on day 1 to the APIH-maintained whole organ cultures. Controls included the sense (s) and one random (r) oligonucleotide. As shown in Fig. 3 B, a 5-d treatment with the as-oligonucleotide almost completely blocked MDGI expression in MEC if compared with the untreated control gland (Fig. 3 A). Wholemount stains of two pairs of contralateral glands (Fig. 3, C and D) demonstrate the typical morphological effect observed upon as-oligonucleotide treatment. Glands placed on the right side of Fig. 3, C and D, received as-oligonucleotide, whereas left glands represent the respective control glands. As can be seen, inhibition of MDGI expression leads to prevention of alveolar end bud formation and to the appearance of glands that seem to be retarded in development. The two other control glands, which received the s-oligonucleotide ( Fig. 3 E, right gland) or r-oligonucleotide (Fig. 3 F,  right side) show the same normal development as the corresponding untreated glands, depicted on the left side in Figs. 3, E and E As could be expected, the appearance of less developed glands was accompanied by a fourfold increase in the labeling index of ductai and ductular epithelial cells (not shown). In accord with impaired alveolar growth shown in Fig. 3, C and D, we found profoundly decreased/~-casein levels in as-oligonucleotide-treated glands (Fig. 3 H), if compared to the normal control (Fig. 3 G). This was particularly evident for the luminal species of alveoli, compared to the intracellular/3-casein level, which was somewhat less affected.

MDGI and EGF Are Functional Antagonists
We showed recently that EGF strongly suppresses MDGI mRNA expression and prevents differentiation in explant cultures derived from virgin or pregnant mouse mammary glands (6,59). It was, therefore, obvious to ascertain whether EGF and MDGI might functionally interact during growth and differentiation. To this end, organ cultures were treated for 5 d with both factors in a concentration range of 1-3 nM (Fig. 4). As shown, EGF exerts mitogenic activities and suppresses the mRNA levels for WAP, ~casein, and MDGI. The effects of EGF on growth and differentiation were suppressed with increasing MDGI concentrations. MDGI at 3 nM completely prevented activities of 1 nM EGF. On the other side, stimulation of functional differentiation by MDGI could be strongly reduced in presence of 3 nM EGF.
In conclusion, increasing the EGF or the MDGI concentrations was mutually antagonistic.

The Activities of MDGI Do Not Depend on Fatty Acid Binding and Can Be Mimicked by an ll-Amino Acid Sequence Represented in the COOH terminus of MDGI and Related FABPs
The physiological role of retinoids, eicosanoids, or longchain fatty acids endogenously bound to FABPs is unclear. We, therefore, tested whether bound fatty acids could be directly involved in the biological activities. The first approach was to produce a recombinant FABP form that cannot bind fatty acid. Replacement of 106 Arg by Thr in human H-FABP abolished the capacity of H-FABP to bind oleic acid, however, did not affect its growth inhibitory (Table I) or differentiation stimulating (Table ID activities. This finding supports a mechanism not involving bound fatty acids. Direct evidence was obtained for a functional domain in MDGI that does not require lipid binding by testing various synthetic peptides of the COOH-terminal MDGI sequence. By comparing FABP sequences encoded by exon 4, a striking homology was detected for a COOH-terminal stretch of 11 amino acids (designated as P108) (Fig. 5 A). Notably, we have shown earlier that P108 does not bind fatty acids (72). P108 at 10 -1° M inhibited DNA synthesis of primary MEC by 55 % (not shown), and was therefore further tested in the organ culture system under conditions as described before for MDGI (Fig. 5 B, a-d). Analogous to MDGI, treatment of APIH-cultured mammary glands for 5 d with 10 -9 M P108 affected ductal branching and caused appearance of more bulbous alveolar end buds (Fig. 5 B, a  and b). In accordance with the morphological changes that suggest suppression of ductal growth by P108, we found that after 3 d, DNA synthesis in MEC was inhibited by •60% (Fig. 5 C). Prolonged treatment for 5 d of the mammary glands with 10 -9 M P108 resulted in the appearance of differentiated epithelium characterized by monolayered, secretory active alveoli (Fig. 5 B, c and d). In parallel, and similarly to MDGI t-casein synthesis was clearly stimulated by P108 (not shown). The growth inhibitory and differentiation stimulating activities of P108 are summarized in Fig. 5 C. As shown, the 11 COOH-terminal residues of MDGI are sufficient to fully mimic the activities of MDGI, including stimulation of B-casein and WAP expression, as well as autostimulation of endogenous MDGI gene expression. Single replacements of 130Lys by Asn or of 128Tyr by Ala abolished the activities of P108. Two other peptides including a shorter sequence of P108 (residues 126-130), representing a functional domain of growth hormone (42), were inactive.

D i s c u s s i o n
We demonstrate here for the first time that a polypeptide growth inhibitor promotes morphological and functional differentiation in the developing nmnunary gland. The in vitro model we have been using ensures to follow ductal growth and branching, lobuloalveolar development, and functional differentiation, i.e., developmental stages normally occurring during pregnancy. We were, therefore, able to test for both growth and differentiation activities of MDGI. It turned out that MDGI, which is expressed in the mammary gland in a developmentally and hormonally regulated manner (6,38,43), specifically inhibits epithelial DNA synthesis, promotes formation of alveolar buds, supports development of differentiated lobuloalveoli, and stimulates milk protein synthesis and fat droplet accumulation. This means that MDGI stimulates both "early" and "late" phases (more characterized by fat droplet accumulation) of functional differentiation. These are all the properties that distinguish MDGI from TGF-/31, which strongly inhibits ductal outgrowth in virgin mice; however, it does not affect epithelial growth during later stages of development like pregnancy. Moreover, contrary to MDGI, TGF-/31 does impair functional differentiation in vitro (41,50) and in vivo (34). Therefore, MDGI can   (7) with sequences of bovine brain FABP (52), bovine heart FABP (5), bovine myelin P2 (68), mouse P422 (68), and bovine CRABP (68). Areas containing identical or conservatively exchanged residues are shadowed. Residues 126-131 are identical to residues 108-112 of bovine growth hormone (42), and they are bordered by two highly conserved positively charged amino acids. Biologically active P108 comprises residues 12M 31 in which cysteine has been replaced by serine to prevent dimerization and inactivation (72). Numbering is according to the MDGI sequence in (7). For comparison, sequences of inactive bovine L-FABP and rat I-FABP are included. (B) Effects of P108 on morphology and histology. Glands were cultured for 5 d in APIH medium in the absence (a and c) or be considered as a unique and novel differentiation factor for the mammary gland.
Functionally, MDGI and the bovine and human forms of H-FABP can not be distinguished. Considering that at least one other member of the family, the brain FABP, has been described to be identical with MDGI (52), one can assume a role for these proteins in tissue differentiation. This assumption concurs with data from an earlier study (73), where we have shown that teratocarcinomas formed in syngenie mice after injection of pluripotent embryonic stem cells treated with MDGI or P108 contain more differentiated neural tissue.
Interestingly, an immunohistochemical analysis of MDGI and H-FABP expression shows immunoreactivity exclusively in postmitotic, terminally differentiated cells of the myocard, skeletal muscle, and surface epithelial cells of intestinal and respiratory tracts (Zschiesche, W., A. Kleine, J. H. Veerkamp, and J. Glatz, manuscript submitted for publication). The developmental, cell and tissue specific expression of various other FABPs (14,15,28,30) indeed seems to support a more general role for these proteins in growth and differentiation similarly to that described here for MDGI.
MDGI expression can be hormonally induced in organ culture and its synthesis is further stimulated when MDGI or P108 were added. To test for a relationship between endogenous MDGI expression and a function of MDGI during development, we have inhibited MDGI expression in MEC. Our findings about a suppression of alveolar growth and /S-casein synthesis might indicate an intracellular mode of action and again indicate to MDGI as a differentiation factor. This is in correspondence with the specific pattern of gene expression during pregnancy showing MDGI transcripts preferentially in alveolar cells adjacent to the surrounding stroma (38). In contrast to the situation in organ cultures, MDGI can not be induced in MEC grown as monolayers, in-eluding responsive to lactogenic hormones murine cell lines (unpublished data). Single MEC provided with an exogenous basement membrane that can be induced to express /3-casein also do not express MDGI unless they form multicellular structures with luminal spaces (unpublished data). These data and our present findings strongly argue in favor of a role for MDGI at a distinct state of development that is characterized by a decline of ductal growth and is associated with lobuloalveolar morphogenesis and functional differentiation.
It has been recently reported about a structural identity between MDGI and a cysteine protease inhibitor specific for papain, cathepsin B and cathepsin L (74). It is tempting to speculate that the high level of MDGI expression found in the lactating mammary gland and in milk (12), suggests a different function which might rather be associated with lipid transport and metabolism, tissue remodeling, or with preserving milk protein products. Our findings about a dominant presence of MDGI in cells with high steroid metabolism such as testicular Leydig' cells, adrenal cells, or cells of the corpus luteum, might indicate future directions of work (Zschiesche, W., A. Kleine, J. H. Veerkamp, and J. Glatz, manuscript submitted for publication): In breast cancer cells, the antiproliferative activity of MDGI can be antagonized by EGF in a dose-dependent manner (10,23). EGF and TGF-c~ strongly suppress MDGI mRNA expression in explant and organ cultures of pregnant or virgin mice (6,59). On the other hand, several lines of indirect evidence indicate that EGF might have a role in differentiation. The levels of EGF in plasma (46.) and its receptor in the mammary gland (20) increase during pregnancy. Both EGF and TGF-ot have been detected in the mammary epithelium of pregnant mice (55,59). However, conflicting reports about EGF or TGF-t~ activities demonstrated both differentiation-inhibiting (31,64,71) and-stimulating activities (71). According to our data, EGF is able to produce a growth stimulatory effect on MEC under conditions of functional differentiation, provided that MDGI was absent or added at low concentrations. The data show that increasing the EGF or MDGI concentration was mutually antagonsitic. We have shown earlier that EGF suppresses MDGI mRNA expression (6), suggesting that under physiological conditions, concentration of both factors might be critical for maintaining balanced growth and differentiation. In early pregnancy, when EGF and TGF-a are present, they could suppress MDGI overexpression, thus preventing impaired growth and differentiation of the mammary gland. Likewise, MDGI could regulate EGF activities to maintain normal differentiation.
Our findings about growth inhibitory and differentiation promoting activities of human and bovine H-FABP provide first direct evidence for a regulatory function of members of this conservative multigene family. Based on expression data, it has been suggested that some FABPs might have in the presence (b and d) of 10 -9 M of the synthetic peptide P108. Whole mount stain of P108 treated gland shows bulbous alveolar end buds (b). Histology shows monolayered secretory active epithelium upon peptide treatment (d). Bar, 50/~m. (C) Peptide P108 fully mimics growth and differentiation properties of MDGI. Whole mammary glands were cultured for 3 d to determine the labeling index in MEC, or for 5 d (to estimate the mRNA levels for MDGI, WAP and/S-casein) in the APIH medium in presence of 1 nM of the different peptides. To verify specificity of P108, LIs were determined with modified peptides: 130Lys-'*130Asn, designated as P206; 128Tyr-*128Ala, designated as P207. In addition, amino acids 69-78 and 126-130 of the MDGI sequence are represented by P14 and P104, respectively. some function in cellular growth, differentiation, and signal transduction (21,69). For the I-FABP, endogenous expression in cryptal cells of the intestinum is strictly associated with inhibition of DNA synthesis (15). However, a dominant expression in more differentiated cells described for the I-FABP (15), the adipocyte FABP, (also referred to as ap2 or P422 [57]), or for the CRABP (14) has not been directly related to growth inhibitory activity. Unexpectedly, transfection of liver eDNA into hepatoma cells (36) or fibroblasts (33) cause these cells to grow in response to linoleic acid and to reach higher cell densities, respectively. Overexpressing CRABP-1 in F9 teratocarcinoma cells resulted in reduction of differentiation (11). However, there are no data available to show the consequences of overexpression of FABPs into normal cells, which can be induced to undergo differentiation in vitro. We report here that I'108, which represents the COOH terminus of MDGI, fully mimics the MDGI properties. This peptide has a strong homology with the COOH terminus of other FABPs, including myelin P2, which is expressed during differentiation of Schwann's cells (28), with CRABP and brain FABP. PI08 does not bind fatty acids (72) rendering involvement of endogenously bound ligands in the activities we described here very unlikely. Rather, our data argue in favor of a mechanism implying interaction of P108 with cellular targets related to growth signaling pathways. For the CRABP, a function in signaling pathways has been suggested (19).
Although the molecular mechanism(s) by which MDGI or P108 inhibit growth and promote differentiation are not yet known, preliminary data indicate steps likely to be involved. We have shown that PI08 over a concentration range of 10 -~° to 10 -m: M prevents arachidonic acid or 15S-hydroxyeicosatetraenoic acid-induced hypersensitivity of neonatal rat heart cells to /3~-adrenergic stimulation (72). From these studies, we concluded that a mechanism of interference of MDGI or P108 subsequent to generation of 15S-hydroxyeicosatetraenoic acid is involved. Among the different peptides tested, only P108 turned out to be active in MEC and heart myocytes. Therefore, it seems possible that the growth modulatory effects of MDGI and of P108 may result from an interference with some pathway subsequent to the generation of lipoxygenase products. For TNF (25), IFN-o~ (26), and TGF-/~ (45), involvement of the arachidonic acid pathway has been documented.