Cadmium exposure activates the ERK signaling pathway leading to altered osteoblast gene expression and apoptotic death in Saos-2 cells
Recent reports of cadmium in electronic waste and jewelry have increased public awareness regarding this toxic metal. Human exposure to cadmium is associated with the development of osteoporosis. We previously reported cadmium induces apoptosis in human tumor-derived Saos-2 osteoblasts. In this study, we examine the extracellular signal-regulated protein kinase (ERK) and protein kinase C (PKC) pathways in cadmium-induced apoptosis and altered osteoblast gene expression. Saos-2 osteoblasts were cultured in the presence or absence of 10 \xce\xbcM CdCl2 for 2\xe2\x80\x9372 hours. We detected significant ERK activation in response to CdCl2 and pretreatment with the ERK inhibitor PD98059 attenuated cadmium-induced apoptosis. However, PKC\xce\xb1 activation was not observed after exposure to CdCl2 and pretreatment with the PKC inhibitor, Calphostin C, was unable to rescue cells from cadmium-induced apoptosis. Gene expression studies were conducted using qPCR. Cells exposed to CdCl2 exhibited a significant decrease in the bone-forming genes osteopontin (OPN) and alkaline phosphatase (ALP) mRNA. In contrast, SOST, whose protein product inhibits bone formation, significantly increased in response to CdCl2. Pretreatment with PD98059 had a recovery effect on cadmium-induced changes in gene expression. This research demonstrates cadmium can directly inhibit osteoblasts via ERK signaling pathway and identifies SOST as a target for cadmium-induced osteotoxicity.
Introduction<!>2.1 Cell culture<!>2.2 Cell treatment<!>2.3 Cell viability assay<!>2.4 Western blot analysis<!>2.5 Apoptotic assay<!>2.6 Real-time PCR<!>2.7 Statistical analysis<!>3.1 Cadmium treatment decreases viability in Saos-2 cells<!>3.2 Cadmium exposure leads to ERK activation but not PKC\xce\xb1 activation<!>3.3 Pretreatment with the ERK inhibitor PD98059 protects against cadmium-induced apoptosis<!>3.4 Cadmium alters mRNA expression of key osteoblast genes<!>3.5 ERK activation participates in cadmium-induced altered osteoblast gene expression<!>Discussion
<p>Cadmium is a heavy metal and widespread environmental contaminant that has gained public attention due to the world-wide increase in discard of electronic waste (e.g., cell phones and computers) containing this toxic metal (Guo et al., 2010; Leung et al., 2008; Wang et al., 2010; Zheng et al., 2008). Because of continued demand for cadmium by industry, this heavy metal continues to be leached into the environment and ultimately accumulates in exposed organisms because of its long biological half life of 10–30 years (Järup and Akesson, 2009).</p><p>Humans are exposed to cadmium through the ingestion of contaminated food or water, smoking cigarettes, or occupational use (Järup and Akesson et al., 2009). Recent reports of cadmium's use in children and adult jewelry highlight other possible routes of human exposure (JAMA, 2010; O'Callaghan, 2010; Weidenhamer et al., 2011). Cadmium intoxication is known to negatively impact human health and the skeleton is a target site for cadmium's toxic action (Järup and Alfven, 2004; Jin et al., 2004; Trzcinka-Ochocka, 2010). One noteworthy case of cadmium-induced advanced bone disease occurred in Japan in the 1940's which led to an outbreak of Itai-Itai disease in women living in a cadmium-polluted region (Tsuchiya, 1978). Over past decades, global epidemiological research indicates that even chronic, low-level exposure to cadmium leads to increased risk of bone fractures and osteoporosis in humans (reviewed by Bhattacharyya 2009; Järup and Alfvén, 2004; Jin et al., 2004) and these findings are confirmed by animal model experiments (Brzóska 2010; Brzóska and Moniuszko-Jakoniuk, 2004, 2005; Regunathan et al., 2003). For example, Brzóska et al. (2005) treated the drinking water of Wistar rats with 1–50 μg/ml for up to 12 months. At all concentrations, cadmium exposure altered bone formation and disrupted the balance between bone formation and resorption, resulting in an osteopenic-phenotype. Together, these studies demonstrate that bone is a critical target of cadmium-induced toxicity.</p><p>There are two proposed mechanisms by which cadmium is able to disrupt bone function. One mechanism is indirect, whereby cadmium damage to kidney or gastrointestinal organs produces a secondary effect on bone (Kjellström, 1992). Alternatively, cadmium can act directly on osteoclasts or osteoblasts by stimulating bone resorption or inhibiting bone formation, respectively (Bodo et al., 2010; Coonse et al., 2007; Regunathan et al., 2003; Smith et al., 2009). We previously reported that cadmium exposure can directly impact osteoblasts by inducing apoptotic death via caspase-3 activation (Coonse et al., 2007). Since apoptosis is an integral component of bone remodeling, disruption of the apoptotic signaling cascades in osteoblasts may contribute to net bone loss, leading to a bone disease state (Xing and Boyce, 2004). The current study aims to further elucidate the mechanisms by which cadmium impacts osteoblasts by examining two cellular pathways and their possible link to apoptotic death and altered gene expression.</p><p>A variety of signaling cascades are implicated in cadmium's ability to disrupt cellular function, including apoptosis (reviewed by Thévenod, 2009). Two signaling pathways known to become activated in response to cadmium exposure in various cell types are the protein kinase C pathway (PKC) and extracellular signal-regulated protein kinase (ERK) which is part of the larger mitogen activated protein kinase (MAPK) family (Chen et al., 2008; Kalariya et al., 2009; Låg et al., 2005; Mantha and Jumarie, 2010; Martin et al., 2006, 2009). Traditionally, ERK is generally considered a cell proliferation pathway with an ability to protect cells against apoptosis (Martin et al., 2009; Santos et al., 2007). However, recent studies indicate ERK activation can lead to cell death signaling (Kalariya et al., 2009; Martin et al., 2006, 2009, 2010; Rasola et al., 2010). Martin et al., 2006 reported that cadmium exposure leads to sustained ERK activation for up to several days and eventually apoptosis through the activation of caspase-3 and caspase-8 in HEK293 human embryonic kidney cells. Iryo et al., 2000 demonstrated that inhibiting the ERK pathway reduced cadmium-induced apoptosis in CCRF-CEM human T lymphoblastoid cells. Other studies link ERK activation to the promotion of apoptosis depending on the cell type and nature of the stimuli (Kim et al, 2005; Rai et al., 2010); and it is significant to point out that none of these studies were done in bone. In addition to the MAPK family, cadmium exposure has been linked to the PKC pathway, where activation has been demonstrated in rat lung and human derived-kidney cells (Låg et al, 2005; Martin et al., 2009). Furthermore, PKC has been shown to negatively regulate sustained ERK activation in HEK cells, causing activated ERK to be down regulated when phosphorylated PKC is present (Martin, et al. 2009).</p><p>The ERK and PKC pathways orchestrate many cellular functions beyond cell death and survival including playing a role in the complex regulation of osteoblast formation, maturation, and differentiation by either repressing or stimulating expression of key osteoblast genes (Addison et al., 2007; Chen, 2003; Delannoy et al., 2001; Nakura et al., 2011; Prouillet et al., 2004; Zhang et al., 2010). We previously reported that cadmium leads to a decrease in mRNA of the runt-related transcriptional factor and a marker for osteoblast differentiation, RUNX2, in cultured Saos-2 cells (Smith et al., 2009). Our results were later confirmed in studies utilizing osteoblasts derived from human knees (Bodo et al., 2010). Other osteoblastic genes reported to decrease in response to cadmium exposure include type I collagen, osterix, bone sialoprotein, ostecalcin and ALP (Bodo et al., 2010; Chen et al., 2009; Iwami and Moriyama, 1993). There is evidence suggesting a role for ERK or PKC signaling the regulation of ALP, SOST, OPN and RUNX2 mRNA (Chen et al., 2003; Jeong et al., 2010; Kono et al., 2007; Lin et al., 2011; Nakura et al., 2011; Prouillet et al., 2004; Vincent et al., 2009). In this study, we examine the bone promoting genes ALP and OPN, as well as the SOST gene whose protein product sclerostin is known to inhibit bone formation (Gallagher and Sai, 2010; Kono et al., 2007; Li et al., 2008). The SOST gene is of particular interest since to date it has not been identified as a target gene in cadmium toxicity, and is currently being evaluated for the treatment of osteoporosis (Gallagher and Sai., 2010, Martin et al., 2008). Although the ERK and PKC pathways appear to be critical regulators in osteoblast function, their roles remain to be determined in cadmium-induced osteotoxicity.</p><p>This research builds upon our previous reports (Coonse et al., 2007; Smith et al., 2009) and by others (Bodo et al., 2010; Lévesque et al., 2008) by examining the signaling pathway involvement leading to osteoblast apoptotic death and altered gene expression. Ultimately, our goal is to advance the current understanding of how environmental toxins contribute to the pathogenesis of osteoporosis by studying the underlying mechanisms involved in cadmium-induced osteotoxicity.</p><!><p>The human osteosarcoma Saos-2 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA). Saos-2 cells were cultured in McCoy's 5A medium (ATCC, Manassas, VA) or Opti-MEM serum free medium (Invitrogen, Carlsbad, CA). McCoy's 5A culture medium was supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Sigma–Aldrich, St. Louis, MO). Opti-MEM culture medium was supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin only. Cells were cultured at 37 °C in air containing 5% CO2. For routine maintenance, medium was changed every 3–4 days and cells were subcultured weekly.</p><!><p>Cells were plated at different densities depending on the assay. Culture medium was changed and treatment was initiated with CdCl2 (Sigma-Aldrich, St. Louis, MO) 24 hr after plating. Cells were treated with 2.5–100 μM CdCl2 for 2–72 hr, which is within the concentration range and exposure time reported in the literature (Pulido and Parrish, 2003). Inhibitor experiments included pretreatment with 5μM of the MEK inhibitor, PD98059 solubilized in DMSO, which blocks the ERK 1/2 pathway or 0.1 μM of the PKC inhibitor, Calphostin C, solubilized in DMSO. Culture medium in inhibitor studies contained less than 0.01% DMSO vehicle (Sigma-Aldrich, St. Louis, MO). Treatment with 5 μM PD98059 or 0.1 μM Calphostin C were determined not be to cytotoxic using an MTT assay for cell viability (data not shown). After treatment, cultures were terminated and adherent cells were collected for evaluation.</p><!><p>Cells were plated at a density of 1×105 cells/well in a 96-well culture plate in McCoy's 5A medium. Cells were allowed to adhere and then treated with cadmium in either McCoy's 5A medium or Opti-MEM medium. After treatment, cells were washed with phosphate buffer saline (PBS) and incubated at 37°C with 20 μg/ml MTT (3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide; ATCC, Manassas, VA) for 4 hr. The conversion of tetrazolium salt MTT to a colored formazan by mitochondrial dehydrogenase was used to assess cell viability. After the supernatant was removed, 100 μl of DMSO was added to each well and absorbance was read at 570nm.</p><!><p>Cells were plated at 6×105 cells/well in a 6-well culture plate. After treatment, cells were lysed and protein concentration was determined using a Bradford assay (Bradford, 1976). Equivalent amounts of protein (40μg) were electrophoresed on a 12% SDS gel and transferred onto PVDF membranes (Bio-Rad, CA, USA). Membranes were blocked in TTBS containing 5% nonfat dry milk for 1 hr and washed three times for a total of 30 minutes with TTBS. Membranes were incubated overnight at 4°C with primary antibodies (Santa Cruz, CA, USA) for phosphorylated ERK (pERK), total ERK (ERK), phosphoylated PKCα (pPKC) or total PKCα (PKC), followed by a 2 hr incubation at room temperature with HRP antibody (Santa Cruz, CA, USA). Immunoreactive protein was detected by exposing the membranes to Immun-Star HRP Chemiluminescent (Bio-Rad, CA, USA), visualized using Quantity One 1-DAnalysis Software, and quantified using Image J software.</p><!><p>The APOPercentage dye (Biocolor, Carrickfergus, UK) was used to detect apoptosis. The dye is transported into an apoptotic cell during the translocation of phosphatidylserine from the inner leaflet to the outer leaflet of the cell membrane. Cells were plated at 1 × 105 cells/well in a 24 well plate. After treatment, cells were washed with PBS, trypsinized off the plate, and collected for a total cell count. The remaining cell suspension was centrifuged and then received APOPercentage dye in fresh medium without serum. The cells were incubated at 37°C for 1 hr washed twice with PBS, and the APOPercentage dye releasing reagent was added. Absorbance was read at 550 nm. Absorbance values were standardized per total cell number. For visual representation, cells were grown on cover slips at 6 × 105 cells/well in a six-well culture plate. After treatment cells were washed with PBS and then received APOPercentage dye in fresh medium without serum, incubated for one hour followed by visualization using a Nikon epifluorescense Eclipse E400 microscope. Digital images were captured using ImagePro software by media Cybergenetics (Silver Spring, MD).</p><!><p>Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA) and quantified at 260nm. Using AffinityScript QPCR cDNA Syntesis Kit (Stratagene, La Jolla, CA), 500 ng of RNA from each sample was reverse transcribed. Real-time PCR was performed using a SYBR ® Green Q-PCR kit (Qiagen, Valencia, CA) Sequences for ALP (5′-TGCAGTACGAGCTGAACAGGA-3′ and 5′-TCCACCAAATGTGAAGACGTG-3′), SOST (5′-CACCACCCCTTTGAGACCAA-3′ and 5′-GGTCACGTAGCGGGTGAAGT-3′), OPN (5′–ACACTGGGCTATGGAGAGGA-3′ and 5′-CTGCCTCTGTGCTGTTGGTA-3′) (Integrated DNA Technologies Inc., Coralvill, IA. All values were normalized using the housekeeping gene, GAPDH (5′-GAGTCCACTGGCGTCTTCAC-3′ and 5′-GGTGCTAAGCAGTTGGTGGT-3′). The PCR conditions were 95°C for 15 minutes, then 40 cycles of 94°C for 15s, 55°C for 30 s, 72°C for 30s. Relative gene expression was determined using a modification of the Pfaffl method (Pfaffl, 2001).</p><!><p>Data represents the mean ± SEM for at least three separate experiments. Data were analyzed using a one-way analysis of variance followed by a Tukey test for multiple comparisons or by a Student's t-test for comparison between two groups. A p-value < 0.05 was considered significant.</p><!><p>Cells cultured in serum-free medium were more sensitive to cadmium toxicity compared to cells cultured in 10% FBS with an EC50 value at 24 hr of 7 μM compared to 117 μM, respectively (Fig. 1A and B). While the use of reduced or serum-free culture medium lowers the CdCl2 concentration needed to elicit a response, serum deprivation can induce or enhance apoptosis (Lopez et al., 2003; Smith et al., 2009) and therefore experiments were conducted using 10% FBS containing culture medium. Based on the results of this study and previous work (Smith etl. al., 2009), subsequent experiments were conducted using 10 μM CdCl2.</p><!><p>In order to assess whether cadmium exposure leads to PKCα activation, cells were exposed to 10 μM CdCl2 for 2–4 hr and levels of pPKCα and total PKCα were evaluated by Western blot. We did not observe activation of PKCα at any of the time points evaluated (Fig 2A and 2B). In contrast, exposure to 10 μM CdCl2 significantly induced phosphoylated ERK at 3, 4, and 18 hr compared to untreated cells (Fig. 3A and 3B). These results demonstrate that exposure to 10μM CdCl2 does not induce activation of PKCα but does lead to a significant activation of the ERK signaling pathway.</p><!><p>Although the ERK pathway is commonly considered an anti-apoptotic or mitogenic signaling pathway, studies demonstrate a proapoptotic signaling role for ERK (Kalariya et al., 2009; Martin et al., 2006, 2009; Rasola et al., 2010). We previously reported that cadmium induces apoptosis in Saos-2 cells using several apoptotic indicators including annexin V staining and caspase-3 activity (Coonse et al., 2007). In this study, treatment with 10 μM CdCl2 for 48 hr resulted in a significant increase in apoptotic cells compared to untreated controls (Fig. 4B), which was also evident when observing stained apoptotic cells (Fig. 4A). Treatment for 1 hr with PD98059 prior to 10 μM CdCl2 exposure attenuated cadmium-induced apoptosis but did not result in complete recovery to a control level (Fig. 4B). Similar results were obtained at the 72 hr time point (Fig. 4C). In contrast, 1 hr pretreatment with Calphostin C was unable to protect Saos-2 cells from apoptosis induced by 10 μM CdCl2 exposure (Fig. 4D). These results, combined with Fig. 3B, suggest the ERK pathway is one of the main pathways involved in cadmium-induced apoptosis in Saos-2 cells.</p><!><p>We previously reported that cadmium exposure leads to a decrease in the key osteoblast transcriptional factor, RUNX2, in cultured Saos-2 cells (Smith et al., 2009). To further investigate the effect of cadmium on osteoblast gene expression, we utilized qPCR and assessed the expression of three other genes essential for the maturation and maintenance of osteoblasts. Cells treated with 10 μM CdCl2 exhibited a significant decrease in OPN and ALP, which are genes known to promote bone formation and matrix deposition (Fig. 5A and 5B). In contrast, the expression of SOST, a known inhibitor of bone formation, increased in response to exposure 10 μM CdCl2 over time (Fig 5C).</p><!><p>Research indicates that ERK activation can lead to a suppressive effect on osteoblast function (Kono et al., 2007). In order to assess whether cadmium-induced ERK activation precedes alterations in osteoblast gene expression, cells were treated 1 hr with PD98059 prior to 10 μM CdCl2 for 18 h. Pretreatment with the ERK inhibitor attenuated the decrease in OPN and ALP expression induced by CdCl2 exposure (Fig. 6A and 6B). Comparably, the observed increase in SOST mRNA expression was reduced to the level of the untreated control in cells pretreated with the ERK inhibitor (Fig 6C). Taken together, these studies demonstrate that the ERK signaling pathway plays a key role in cadmium-induced apoptosis and alteration of osteoblast genes in Saos-2 cells.</p><!><p>Cadmium has long been known to cause adverse effects on human health, including increased susceptibility to developing metabolic bone disorders such as osteoporosis (Bhattacharyya, 2009; Järup and Alfvén, 2004; Järup and Akesson 2009; Trzcinka-Ochocka et al., 2010; Tsuchiya, 1978). Although cadmium is a recognized toxic metal, little is known about how cadmium directly influences bone-forming osteoblasts.</p><p>The ERK pathway is traditionally known to function in cell proliferation with short-term activation; however, recent studies link sustained activation of ERK to apoptosis (Martin et al., 2006; reviewed by Thévenod 2009). Cadmium exposure leads to activation of the ERK pathway and ultimately apoptosis in several non-osseous cell types (Iryo, et al., 2000; Martin et al., 2006, 2009; Yang et al., 2009). Our results extend these previous studies to include human osteoblasts. We demonstrate that cadmium exposure activates ERK in human-derived Saos-2 osteoblasts acutely (3 and 4 hr) and that the pathway remains active at 18 hr. Furthermore, blocking the ERK pathway with the MEK inhibitor PD98059 protects Saos-2 cells from cadmium-induced apoptotic death. Martin et al., (2006) demonstrated that exposure to 1.25 μM CdCl2 was able to phosphorylate ERK in HEK293 human embryonic cells after 6 days of sustained treatment in culture and resulted in caspase-3 and caspase-8 activation. Involvement of the ERK pathway in cadmium-induced apoptosis in mesangial cells has also been reported (Yang et al., 2009). Additionally, Rai et al., (2010) showed that inhibition of the ERK pathway with PD98059 rescued cells from apoptotic death after the addition of a metal mixture that included cadmium.</p><p>In contrast to the ERK pathway, we were unable to detect PKCα activation in response to cadmium, and blocking the PKC pathway with Calphostin C did not protect cells from cadmium-induced apoptotic death. Our results are consistent with those of Templeton et al., (1998) whose data demonstrate that PKC is not activated in renal mesangial cells after exposure to 5–30 μM CdCl2 at any time point evaluated. Crosstalk between ERK and the protein kinase C (PKC) pathway has also been reported after cadmium exposure (Låg et al., 2005; Martin et al., 2009; Miyahara et al., 2004) and the PKC pathway is known to negatively regulate sustained ERK activation in HEK kidney cells, causing activated ERK to be down regulated when phosphorylated PKC is present (Martin et al., 2009).</p><p>It is noteworthy that although the ERK pathway inhibitor was able to attenuate cadmium-induced apoptosis in Saos-2 cells, it was unable to bring the percent of apoptosis back to a control level, which implies other signaling pathways may be involved in cadmium-induced cell death. Studies indicate that inhibiting p53 or other members of the MAPK pathway including JNK and p38, can block cadmium-induced apoptosis in lung and skin cells (Galán et al., 2000; Lag et al., 2005; Son et al., 2010). Additional reserach will be required to determine the involvement of other signaling pathways in cadmium-induced apoptosis in Saos-2 cells.</p><p>In addition to playing a role in cadmium-induced apoptosis, our research links ERK activation to cadmium-induced alteration in Saos-2 gene expression. Cadmium exposure has been shown to reduce the mRNA expression of several osteoblast genes, such as RUNX2, collagen I, ALP, osterix, and osteocalcin in different in vitro osteoblast culture systems (Bodo et al., 2010; Brzóska et al., 2007; Smith et al., 2009). To further explore the effects of cadmium on osteoblast cells, we focused on the pro-survival, bone promoting genes ALP and OPN, as well as SOST, whose protein product sclerostin inhibits bone formation and is known to induce apoptosis in human osteoblasts (D'Amelio et al., 2010; Denhardt et al., 2001; Li et al., 2008; Thurner et al., 2010; Vincent et al., 2009). Our results indicate that cadmium exposure may contribute to an osteoporotic state by repressing genes that promote bone mineralization (ALP and OPN) while increasing the expression of genes known to inhibit bone formation (SOST). This study distinguishes itself by identifying SOST as a target for cadmium-induced osteotoxicity.</p><p>Research indicates that ERK activation can lead to a suppressive effect on osteoblast function and acts, in part, by regulating key osteoblast genes such RUNX2, ALP, OPN, and type I collagen (Addison et al., 2010; Chen et al., 2003; Kono et al., 2007; Prouillet et al., 2004; Zhang et al., 2010). In this study, we demonstrate that changes in the mRNA expression of ALP, OPN, and SOST induced by cadmium exposure act via activation of the ERK signaling pathway. These results are consistent with other osteoblast studies that show pretreatment with PD98059 recovers the decrease in ALP induced by H2O2 (Bai et al., 2004) and ERK activation leading to increased SOST mRNA expression after exposure to TNF-related weak inducer of apoptosis (TWEAK) in osteoblasts (Vincent et al., 2009). This is the first report linking the ERK signaling pathway to cadmium-induced altered gene expression in osteoblasts.</p><p>In summary, this study provides insight into the mechanisms underlying cadmium-induced osteotoxicity by identifying the ERK pathway as a critical mediator in cadmium-induced apoptosis and changes in Saos-2 osteoblast gene expression. In addition, this study distinguishes itself by identifying SOST as a target for cadmium-induced osteotoxicity. Collectively, our research adds to the current understanding of how environmental toxins, such as cadmium, interfere with osteoblast function and thus may contribute to the pathogenesis of bone disease.</p>