ToxSci Advance Access originally published online on February 16, 2006
Toxicological Sciences 2006 91(1):255-264; doi:10.1093/toxsci/kfj135
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Mono-(2-ethylhexyl) Phthalate Rapidly Increases Celsr2 Protein Phosphorylation in HeLa Cells via Protein Kinase C and Casein Kinase 1
* Division of Biological Sciences, CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709; and Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
1 To whom correspondence should be addressed at CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, NC 27709. Fax: (919) 558-1300. E-mail: kjohnson{at}ciit.org.
Received November 4, 2005; accepted February 13, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phthalates are ubiquitous environmental contaminants that target the fetal and pubertal testis and lead to alterations in endocrine and spermatogenic function. Some features of phthalate-induced testicular injury suggest that phthalates alter Sertoli–germ cell adhesion and G protein signaling. Celsr2 is a unique protein that has structural characteristics of both an adhesion molecule and a G protein coupled receptor (GPCR) and has been demonstrated to function in Sertoli–germ cell adhesion. Within 2 h of a 1-g/kg mono-(2-ethylhexyl) phthalate (MEHP) exposure, in vivo Sertoli cell celsr2 localization was altered; celsr2 immunostaining became concentrated in the basal aspect of Sertoli cells, and then a diffuse pattern emerged. Because GPCRs are regulated by phosphorylation, the hypothesis that phthalate exposure induces the phosphorylation of celsr2 was tested by examining phosphorylation in celsr2-transfected HeLa cells treated with MEHP. At concentrations of 1µM or greater, MEHP transiently increased celsr2 phosphorylation on serine/threonine residues; celsr2 phosphorylation was increased by 15 min of exposure and returned to control levels after 60 min. Cells exposed to the inactive phthalate monoester mono-methyl phthalate showed no change in celsr2 phosphorylation. In addition, phosphorylation of the endogenous HeLa cell GPCR, Chemokine Receptor 4 (CXCR4), was not altered by exposure to MEHP. Inhibition of protein kinase C or casein kinase 1 prevented MEHP-induced celsr2 phosphorylation, while inhibition of protein kinase A or mitogen-activated protein kinase had no effect. These data show that MEHP exposure rapidly alters testicular celsr2 immunolocalization as well as celsr2 posttranslational modification in a model cell line.
Key Words: MEHP; celsr2; GPCR; PKC; CK1.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phthalates are chemicals used as softening agents in plastic-containing products. Phthalates are not covalently bound to the matrix and therefore leach out of the plastic materials over time, resulting in environmental contamination. Since many consumer products contain phthalates including adhesives, detergents, personal care products, and medical devices, human exposure via ingestion or dermal contact is ubiquitous (Silva et al., 2004
). In rodents, high-dose phthalate exposure targets the fetal and pubertal testis, leading to alterations in endocrine and spermatogenic function (Boekelheide et al., 2004; Foster, 2006). Recent research has suggested that in utero phthalate exposure may adversely affect male reproductive development in humans, as evidenced by a correlation between higher maternal phthalate exposure and decreased anogential distance and incomplete testicular descent in male infants (Swan et al., 2005). Because of the potential reproductive and developmental effects of human phthalate exposure, there is a critical need to understand the molecular mechanisms of phthalate-induced testicular injury.
Previous research has suggested that the Sertoli cell is a proximal cellular target during pubertal phthalate-induced testicular injury (Boekelheide et al., 2004). Histological and functional alterations in the Sertoli cells of pubertal rats occur within 3 h of high-dose in vivo phthalate exposure (Creasy et al., 1983; Dalgaard et al., 2001; Richburg and Boekelheide, 1996). Observed effects included displacement of germ cells from the basal epithelium, Sertoli cell vacuolization, and loss of Sertoli–germ cell adhesion resulting in germ cell sloughing into the lumen of the seminiferous tubule (Chapin et al., 1988; Creasy et al., 1983). Although the in vivo phthalate molecular target is unknown, GPCR-based intracellular signaling is affected in a Sertoli cell culture model. Phthalate exposure inhibited follicle stimulating hormone (FSH)–stimulated cAMP production and suppressed basal and FSH-stimulated Sertoli cell proliferation (Heindel and Chapin, 1989; Li et al., 1998; Lloyd and Foster, 1988) but did not compete with FSH binding to its receptor (Chapin et al., 1988; Grasso et al., 1993; Gray and Beamand, 1984). The failure of phthalate to alter FSH binding indicated that the FSH receptor itself may not be a main proximal target. However, the inhibitory effect of MEHP on FSH-stimulated cAMP accumulation suggested that Sertoli cell signaling stimulated by a GPCR is an important phthalate target. On the whole, these data indicate that phthalates alter Sertoli–germ cell adhesion and G protein signaling.
While investigating potential Sertoli–germ cell adhesion proteins, a promising phthalate target protein termed celsr2 was identified. Celsr2 is a Sertoli cell product that has structural characteristics of both an adhesion molecule and a GPCR (Beall et al., 2005; Shima et al., 2004). Celsr2 is composed of nine extracellular cadherin repeats, which indicate a possible cell adhesion function (Johnson et al., 2000), and a seven-transmembrane-spanning motif that can potentially interact with G proteins. A potential role for celsr2 in Sertoli–germ cell adhesion has been demonstrated using cocultures of primary Sertoli and germ cells. The addition of a celsr2 protein fragment consisting of a portion of the extracellular domain resulted in germ cell detachment from Sertoli cells, similar to that observed following coculture treatment with MEHP (Beall et al., 2005). Based on these data, celsr2 is an attractive target for phthalate testicular injury.
Because GPCRs are regulated by protein phosphorylation (Marchese et al., 2003; Morris and Malbon, 1999), we hypothesized that phthalate exposure modifies intracellular signaling events leading to changes in the phosphorylation state of celsr2. Using a cell culture model, our results support this hypothesis by showing that phthalates increase celsr2 phosphorylation, via protein kinase C (PKC) and casein kinase 1 (CK1), within minutes of exposure.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Male Fisher 344 rats (Charles River Laboratories Inc., Wilmington, MA) were used after an acclimation period of 1 week and were treated according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals; all experiments were approved by the Brown University IACUC committee. Rats were housed in plastic cages at constant room temperature (70 ± 2°F) and humidity (30–70%) with a 12-h alternating light-dark schedule and were given water and chow (Pro-Lab Rat, Mouse, and Hamster Chow 3000, St. Louis, MO) ad libitum.
Postnatal day (PND) 28 male rats were exposed via oral gavage to 1 g/kg mono-(2-ethylhexyl) phthalate (MEHP) (TCI America, Portland, OR) suspended in corn oil. Control rats received corn oil alone. At each postexposure time point, three rats were killed by carbon dioxide asphyxiation. Testes were collected at 0, 1, 2, 3, 6, and 12 h postexposure for the preparation of frozen sections.
Cell culture and transfections.
HeLa human cervical cancer cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). Cells were transfected with celsr2 plasmid cDNA fused to a 3X FLAG tag (Met-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr-Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) or fused to enhanced green fluorescent protein (EGFP). The celsr2-FLAG construct was produced as previously described (Beall et al., 2005). Briefly, the full-length mouse celsr2 cDNA was fused to a 3X FLAG tag and ligated into pIRES2-EGFP (Invitrogen Corporation, Carlsbad, CA), in which both celsr2 and EGFP are under the control of a cytomegalovirus (CMV) promoter. The celsr2-EGFP construct was produced by ligating full-length celsr2 into pcDNA3.1+ in which gene expression was under the control of a CMV promoter. EGFP was subcloned from pIRES2-EGFP by PCR amplification using the following primer pairs: forward, 5'-GAT ACG CGT GCC ACA ACC ATG GTG AG-3' and reverse, 5'-GCC GAA TTC CTT GTA CAG CTC GTC CA-3'. The EGFP PCR product was gel purified and ligated in-frame onto the C-terminus of celsr2. The construct was verified by restriction enzyme digestion. HeLa cells were transiently transfected with 1 µg of plasmid DNA using TransIT-HeLa MONSTER (Mirus Bio Corporation, Madison, WI) according to the manufacturer's protocol. The use of HeLa MONSTER resulted in a transfection efficiency of 60%, as demonstrated by EGFP fluorescence. These experiments were carried out in a model cell line rather than in primary Sertoli cells or in a Sertoli cell line due to the difficulty of achieving celsr2 protein expression levels necessary for phosphorylation analysis in Sertoli cells (data not shown).
Treatment.
The phosphorylation of celsr2 in response to phthalate treatment was examined by starving subconfluent cultures of HeLa cells in serum- and phosphate-free DMEM (GibcoBRL Inc., Grand Island, NY) for 4 h to reduce the basal levels of phosphorylation. Following starvation, the cells were stimulated with FCS in the presence of 100µM of the active phthalate congener MEHP, the nonactive congener mono-methyl phthalate (MMP), or vehicle (0.1% dimethylsulfoxide [DMSO]) for various time lengths. 100µM MEHP was chosen as the experimental concentration because it is the highest concentration that does not cause cytotoxicity (Phillips et al., 1986) and approximates in vivo testicular phthalate concentrations after gavage exposure of 10–100 mg/kg phthalate diester (Fennell et al., 2004). The phthalate congeners are described as active or nonactive based on their toxicity in the rodent male reproductive system (Creasy et al., 1983; Gray et al., 2000; Heindel and Chapin, 1989). To examine the role of specific kinases in the phosphorylation of celsr2, the cells were pretreated with chemical kinase inhibitors for 20 min prior to MEHP exposure. Several kinase inhibitors were tested: (1) 20µM PD98059, an Erk mitogen-activated protein kinase (MAPK) inhibitor; (2) 150nM H89, a protein kinase A (PKA) inhibitor; (3) 20µM IC261, a CK1 inhibitor; and (4) 1µM GF109203x or 1µM G06983
[GenBank]
, PKC inhibitors.
Immunofluorescence—HeLa cells.
After MEHP or DMSO treatment, HeLa cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Nonspecific binding was blocked with 5% normal goat serum, and the cells were incubated with 1 µg/ml FLAG M2 monoclonal antibody (Sigma Aldrich, St. Louis, MO) for 2 h at room temperature in a humidified chamber. Immunoreactivity was detected using an Alexa488-conjugated secondary antibody (Molecular Probes, Eugene, OR). Negative controls were incubated without primary antibody or with normal mouse IgG.
Celsr2 localization—HeLa cells.
HeLa cells were transfected with celsr2-EGFP and exposed to 10µM MEHP, 100µM MEHP, or DMSO for 0, 15, 30, or 45 min, and celsr2 was subsequently localized using a Zeiss Axiovert fluorescent microscope (Carl Zeiss MicroImaging, Thornwood, NY). The cells were exposed to MEHP in CO2-Independent medium (GibcoBRL Inc.) and kept at 37°C using the Tempcontrol 37-2 digital temperature regulator and a heated mounting frame (Carl Zeiss MicroImaging).
Celsr2 localization—rat testis.
Celsr2 antibody production, testicular immunostaining specificity, and the testicular immunostaining protocol have been described previously (Beall et al., 2005). Testis cryosections from MEHP-treated and control animals were flash-frozen in optimum cutting temperature compound (Sekura Finetek Inc., Torrance, CA) using liquid nitrogen, and 8 µm cryosections were fixed in –20°C acetone for 5 min. After blocking for 30 min with PBS+ (5% normal goat serum, 0.1% bovine serum albumin in phosphate-buffered saline [PBS], pH 7.4), sections were incubated with a 1:20 dilution of anti-celsr2 antibody overnight at 4°C. After washing with PBS, sections were incubated for 45 min at room temperature with a 1:500 dilution of goat anti-chicken IgG coupled to Alexa594 (Molecular Probes). After washing with PBS and mounting with GelMount (Biomeda Corp., Foster City, CA), sections were viewed using a Zeiss Axiovert 35 microscope, and images were captured with a Spot RT digital camera (Diagnostic Instruments Inc., Sterling Heights, MI).
To quantify the altered celsr2 immunostaining pattern, sections from control and MEHP-exposed rats at each time point were analyzed blindly. All tubules in a cross section were scored into one of three groups based on the predominant staining pattern (> 50%) in each seminiferous tubule. Seminiferous tubules that displayed a Sertoli cell (spoke-like) celsr2 immunostaining pattern reaching from the basal epithelium toward the lumen were classified as spoke-like. Seminiferous tubules that lost the spoke-like pattern and displayed celsr2 immunostaining localized in a dense circular pattern within the basal epithelium were categorized as exhibiting basal condensation. Loss of celsr2 immunostaining so that the seminiferous tubules showed only background staining was characterized as no staining. The number of tubules in each category was divided by the total number of tubules scored, and this percentage was analyzed for statistical significance.
Immunoprecipitation and Western blot analysis.
Cell lysates were prepared in phosphosafe extraction buffer (EMD Biosciences, San Diego, CA) containing complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). The supernatants obtained after centrifuging the samples at 12,000 x g for 15 min at 4°C were used for immunoprecipitation and subsequent Western blot analysis. Protein concentration was measured using the bicinchoninic acid assay (Pierce, Rockford, IL). Samples containing 300 µg of total protein were incubated with 1 µg of anti-FLAG M2 monoclonal antibody (Sigma Aldrich) for 2 h at 4°C with constant rotation. To capture the immune complexes, 20 µl of immobilized protein A/G (Pierce) was added to the samples, and the samples were rotated overnight at 4°C. The immune complexes were washed three times in 0.5 ml of ice-cold wash buffer (0.05M Tris, pH 7.4, and 0.15M NaCl) and released from the beads by boiling for 5 min in 25 µl of 2x sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. The immunoprecipitates were fractionated by SDS-PAGE, transferred to PolyVinylidine DiFluoride Immobilon-P membrane (Millipore Corporation, Bedford, MA), and the nonspecific binding sites were blocked with Tris-buffered saline, pH 7.4, containing 2% nonfat dry milk. The membranes were then incubated with a 1:400 dilution of monoclonal anti-phosphothreonine antibody and a 1:2000 dilution of monoclonal anti-phosphoserine antibody (Sigma Aldrich) overnight at 4°C with gentle agitation. Immunoreactivity was detected using a 1:25,000 dilution of mouse IgG, horseradish peroxidase–linked whole antibody (Amersham Biosciences Corp., Newark, NJ), Enhanced Chemiluminescence Plus (Amersham Biosciences Corp.), and the Alpha Innotech image station (Alpha Innotech Corporation, San Leandro, CA). The blots were stripped in 7M guanidine hydrochloride and reprobed with a 1:5000 dilution of anti-FLAG M2 monoclonal antibody. Immunoreactivity was quantified by densitometry using the Alpha Innotech software, and the ratio of phosphorylated to total protein in the control samples was set to 1.
Statistical analysis.
Data depicted in the phosphorylation graphs represent the mean ± standard deviation of results obtained from three to six replicates. Each replicate was an average of three wells. Intergroup comparisons were made using ANOVA with the Dunnet post hoc significance test. Statistical significance in tubule quantification was determined by differences (p < 0.05) between the groups detected using a one-way ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phthalate Increases Celsr2 Serine/Threonine Phosphorylation
The investigation of a potential phthalate-induced effect on celsr2 phosphorylation was begun using subsequence analysis of the celsr2 protein to predict potential phosphorylation motifs located in the C-terminus and in the intracellular loops. The majority of the potential phosphorylation motifs corresponded to serine/threonine kinases, including 16 potential PKC motifs, 9 potential PKA motifs, 3 potential MAPK motifs, 10 potential CK1 motifs, and 11 potential CK1/CK2 motifs (Xue et al., 2005
; Zhou et al., 2004). Because the majority of potential phosphorylation motifs contained serine or threonine, experiments were conducted to determine whether celsr2 was phosphorylated on serine/threonine residues and whether phosphorylation was altered by exposure to MEHP.
The effect of phthalate exposure on celsr2 phosphorylation was examined by transiently transfecting HeLa cells with FLAG-tagged celsr2 cDNA. The expression of celsr2 was verified by Western blot analysis with a FLAG antibody and a celsr2 antibody (Beall et al., 2005). As expected, both full-length and 60-kDa membrane/cytoplasmic fragments were observed in transfected but not untransfected cells (data not shown). The 60-kDa fragment was predicted by sequence homology to other GPCRs containing a juxtamembrane region termed the GPCR proteolysis site (Beall et al., 2005; Krasnoperov et al., 2002; Usui et al., 1999). Since the 60-kDa fragment is likely the mature form present at the cell surface (Krasnoperov et al., 2002), further analysis of celsr2 phosphorylation was performed by examining this fragment.
Phthalate effects on celsr2 phosphorylation were examined in transfected cells by comparing MEHP exposure to either MMP or vehicle (0.1% DMSO) exposure. Comparison of untreated and DMSO-treated cells demonstrated that DMSO did not alter celsr2 phosphorylation (data not shown). Treatment of transfected cells with 100µM MEHP rapidly increased the serine/threonine phosphorylation of celsr2. MEHP-induced effects were seen after 15 and 30 min of phthalate exposure. Phosphorylation returned to control levels by 60 min of MEHP exposure (Fig. 1). MEHP had no effect on the amount of total celsr2 protein present in the cells (data not shown). Unlike MEHP, exposure to MMP failed to alter celsr2 serine/threonine phosphorylation (Fig. 2).
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celsr2 protein subsequence analysis also identified a number of potential tyrosine phosphorylation motifs corresponding to various growth factor receptor consensus sites (Xue et al., 2005
; Zhou et al., 2004). Despite the presence of these sites, 100µM MEHP failed to modify the tyrosine phosphorylation of celsr2 compared to DMSO-treated controls (Fig. 3).
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Phthalate Does Not Change CXCR4 Serine/Threonine Phosphorylation
Since celsr2 contains the characteristic seven-pass transmembrane region of GPCRs, celsr2 is a putative GPCR (Johnson et al., 2000
; Usui et al., 1999). To determine whether the altered celsr2 phosphorylation in response to MEHP was specific to celsr2 or the result of a widespread effect on GPCRs, cell lysates were consecutively immunoprecipitated for celsr2 and Chemokine Receptor 4 (CXCR4). CXCR4 is a GPCR that is endogenously expressed in HeLa cells and is phosphorylated on serine/threonine residues within minutes in response to agonist (Hezareh et al., 2004). The phosphorylation of celsr2 was significantly increased in MEHP-treated cells compared to control cells (Fig. 4A), but MEHP exposure failed to alter the serine/threonine phosphorylation of CXCR4 (Fig. 4B).
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CK1 and PKC but Not MAPK or PKA Play a Role in MEHP-Induced Celsr2 Phosphorylation
The subsequence analysis of celsr2 protein predicted a number of potential phosphorylation sites, including sites for PKC, PKA, MAPK, CK1, and CK1/2 (Xue et al., 2005
; Zhou et al., 2004). Inhibition of CK1 with IC261 failed to alter the basal phosphorylation of celsr2 but prevented the MEHP-induced increase in celsr2 phosphorylation (Fig. 5A). Inhibition of PKC with 1µM GF109203x or 1µM G06983
[GenBank]
also failed to change celsr2 basal phosphorylation but prevented the increase in celsr2 phosphorylation in response to MEHP (Figs. 5B and 5C). Inhibition of CK1 using IC261 and inhibition of PKC using GF109203x also decreased MEHP-induced phosphorylation below control levels (Figs. 5A and 5B). The inhibition of MAPK signaling with 20µM PD98059 (Fig. 6A) or PKA signaling with 150nM H89 (Fig. 6B) did not alter either basal or MEHP-induced celsr2 phosphorylation.
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Phthalate Concentration Response of Celsr2 Phosphorylation
To determine the celsr2 phosphorylation response at lower MEHP concentrations, transfected cells were exposed to 0, 0.1, 1, 10, or 100µM MEHP for 15 min. Celsr2 phosphorylation was significantly increased following exposure to 1, 10, or 100µM MEHP (Fig. 7). No change was observed at 0.1µM MEHP. There was no significant difference in the celsr2 phosphorylation levels between the effective concentrations.
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Celsr2 Localization in HeLa Cells Was Not Altered by MEHP Exposure
Because GPCR phosphorylation may alter its subcellular localization (Marchese et al., 2003
; Morris and Malbon, 1999), the distribution of celsr2 in transiently transfected HeLa cells was examined following MEHP exposure. In cells transfected with the FLAG-tagged celsr2 construct used for phosphorylation studies, the celsr2 distribution pattern was heterogeneous. While some cells showed a predominantly cytoplasmic celsr2 pattern (Fig. 8A, arrowhead), most cells had some cytoplasmic celsr2 but also contained plasma membrane celsr2 localization (Fig. 8A, arrow). Exposure of these transfected cells to MEHP did not appear to change the overall celsr2 immunostaining pattern (data not shown). Because the subcellular distribution of transfected celsr2 was variable among the cultured cells, a potential effect of MEHP on celsr2 subcellular distribution also was examined in individual cells. For these experiments, the localization of celsr2 following MEHP exposure was investigated in cells transfected with celsr2 containing a C-terminal EGFP tag. This construct was used to enable celsr2 localization in live cells. Like the FLAG-tagged celsr2 protein, the celsr2-EGFP protein localized, in most cells, to the plasma membrane with some cytoplasmic staining visible (Fig. 8B). Treatment with 10µM MEHP (data not shown) or 100µM MEHP for 15 (data not shown), 30 (data not shown), or 45 min (Fig. 8C) did not alter the cellular distribution of celsr2-EGFP.
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Celsr2 Localization In Vivo Was Altered by Phthalate Exposure
To determine whether celsr2 was altered in rat testis following high-dose phthalate exposure (1 g/kg), celsr2 protein localization following phthalate-induced testicular injury was examined. Celsr2 localization was examined in PND 28 rat testis 0, 1, 2, 3, 6, and 12 h post–MEHP exposure. In control testis, celsr2 protein localized to the basal epithelium in a spoke-like, Sertoli cell pattern (Fig. 9A) that remained unchanged over time (data not shown). By 2 h after MEHP exposure, the spoke-like Sertoli cell pattern collapsed into basal condensations near the Sertoli cell nucleus (Fig. 9B), and this pattern persisted at 3 and 6 h after exposure (data not shown). By 12 h, celsr2 staining was absent in the majority of tubules (Fig. 9C). Punctate celsr2 immunostaining associated with germ cells was not altered during phthalate-induced testicular injury (data not shown).
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The celsr2 immunostaining pattern was quantified following MEHP exposure. The percentage of seminiferous tubules displaying the normal Sertoli cell spoke-like pattern was significantly reduced at all time points following MEHP exposure (Fig. 10A). The percentage of seminiferous tubules displaying the MEHP-induced basal condensation pattern was increased significantly at 2 h and at all subsequent time points (Fig. 10B). The percentage of seminiferous tubules with no celsr2 immunostaining was significantly increased at 12 h following MEHP exposure (Fig. 10C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using HeLa cells as a model, the major conclusions of this research are the following: (1) celsr2 phosphorylation on serine/threonine residues is increased within 15 min of MEHP exposure; (2) PKC and CK1 are involved in the MEHP-induced phosphorylation of celsr2; and (3) the increased phosphorylation appears to be specific to celsr2 or a subset of GPCRs, including celsr2. Furthermore, our in vivo data demonstrate that celsr2 localization in testis is altered following phthalate exposure.
It was hypothesized that phthalates would induce changes in celsr2 similar to those seen after agonist binding to a model GPCR. Two conserved events (receptor phosphorylation and internalization) occur following agonist binding to GPCRs (Marchese et al., 2003; Morris and Malbon, 1999). This study examined the phosphorylation and localization of celsr2 in response to MEHP. The phosphorylation of celsr2 was examined in a model cell line because no antibodies were available to immunoprecipitate endogenous celsr2, while celsr2 localization was examined in both HeLa cells and rat testis. The study demonstrated that exposure of a model cell line to MEHP, but not a nontoxic phthalate, altered celsr2 phosphorylation. MEHP resulted in a significant increase in the serine/threonine phosphorylation of celsr2 but had no effect on tyrosine phosphorylation. The peak phosphorylation occurred between 15 and 30 min of MEHP exposure and returned to control levels by 60 min. In addition, the altered phosphorylation was specific to celsr2 or possibly a subset of GPCRs and not a global effect, as demonstrated by the failure of MEHP to alter the phosphorylation of an endogenous HeLa cell GPCR (CXCR4). The transient nature of celsr2 phosphorylation is likely an adaptive response, similar to what is observed with changes in gene expression following phthalate exposure (Liu et al., 2005).
Changes in celsr2 phosphorylation occurred at relatively low MEHP concentrations. Our investigation was performed using up to 100µM MEHP. This concentration was chosen because it produced maximal germ cell detachment in Sertoli–germ cell cocultures (data not shown) and altered FSH signaling in Sertoli cells (Grasso et al., 1993). However, the concentration response data demonstrated that MEHP amounts as low as 1µM increased celsr2 phosphorylation. These data are significant because 1µM MEHP is well below the maximal MEHP tissue concentration (
100µM) seen in animals following in vivo DEHP exposure at a level significantly lower than the current LOAEL (Kavlock et al., 2005). Research demonstrated that exposure of Sertoli–germ cell cocultures to 1µM MEHP resulted in germ cell detachment and impaired basal and FSH-stimulated Sertoli cell proliferation 12 h postexposure (Li et al., 1998). This research, combined with our findings, demonstrates that exposure to MEHP at relatively low concentrations results in altered signaling. An obvious caveat to our studies is that the response of the testicular target cell to phthalates may differ from the HeLa cell response. However, these data suggest that altered protein phosphorylation and intracellular signaling may occur in the testis after DEHP exposure at a level significantly lower than the current LOAEL.
These results are some of the first to demonstrate altered protein phosphorylation following phthalate exposure. Phosphorylation of MAPK in primary rat Sertoli cells was decreased by 500µM MEHP to levels below control 20 min after exposure (Bhattacharya et al., 2005). In combination with the phthalate response of celsr2 phosphorylation, these data show that intracellular signaling processes are affected within minutes of phthalate exposure, even at 1µM concentrations. This result raises the possibility that low doses of phthalates may adversely affect cellular homeostasis. Our results demonstrated only a modest alteration in celsr2 phosphorylation. However, even subtle changes in protein phosphorylation may dramatically affect cell signaling (Grubb et al., 2003).
While many GPCRs are phosphorylated by G protein receptor kinases, others are subject to phosphorylation by second messenger–dependent kinases such as PKA and PKC (Marchese et al., 2003; Morris and Malbon, 1999). Amino acid analysis of celsr2 predicted numerous phosphorylation motifs, including many corresponding to PKA and PKC (Xue et al., 2005; Zhou et al., 2004). Because of this, the role of these kinases in the phosphorylation of celsr2 was investigated. It was found that inhibition of PKC, but not PKA, prevented the MEHP-induced phosphorylation of celsr2, suggesting that MEHP results in the activation of PKC. In addition, celsr2 contains potential phosphorylation motifs for CK1 and MAPK (Xue et al., 2005; Zhou et al., 2004); therefore, the role of these kinases was also examined. Inhibition of CK1 prevented the MEHP-induced phosphorylation of celsr2, while inhibition of MAPK had no effect. The inhibition of CK1 and the inhibition of PKC using GF109203x did not affect basal celsr2 phosphorylation but decreased MEHP-induced phosphorylation below control levels. This decrease may be due to the nonspecific inhibition of kinases, such as PKC
and GSK3, which are inhibited by GF109203x, but not G06983
[GenBank]
. Additionally, there may be cross talk between CK1 and PKC that results in the partial inhibition of both pathways following inhibitor treatment. Alternatively, both CK1 and PKC may be involved in the regulation of another as yet unidentified kinase involved in celsr2 phosphorylation.
In most transfected HeLa cells, celsr2 exhibited mainly plasma membrane localization, with some intracellular staining visible. The intracellular staining most likely corresponds to Golgi complex and endoplasmic reticulum–associated celsr2. The localization of celsr2 in the Golgi may represent celsr2 processing and trafficking through the Golgi to the plasma membrane. The distribution of celsr2 in HeLa cells was unchanged after MEHP exposure, suggesting that celsr2 was not internalized quantitatively following phthalate-induced phosphorylation.
The localization of celsr2 was also examined in rat testis. Rat testis cryosections exhibited a spoke-like celsr2 staining pattern characteristic of Sertoli cells and a punctate pattern of presumed germ cell localization (Beall et al., 2005). Within 2 h of MEHP exposure, the spoke-like pattern collapsed into basal condensations that persisted at 3 and 6 h. By 12 h, celsr2 staining was absent in the majority of seminiferous tubules. The germ cell staining pattern was unaffected by MEHP exposure. MEHP is thought to directly target Sertoli cells but not germ cells; therefore, phthalate-induced celsr2 redistribution may occur only in Sertoli cells.
The differential effect of phthalates on celsr2 distribution in Sertoli cells versus HeLa cells may be due to one of several factors: (1) a difference in Sertoli and HeLa cell biology, (2) a consequence of the added GFP tag, or (3) a differential impairment of the basic cytoplasmic structure. The Sertoli cell celsr2 immunostaining pattern is similar to that of the Sertoli cell Golgi complex (Johnson and Boekelheide, 1993). Golgi complex distribution is determined by the cytoskeleton, and phthalate exposure rapidly collapses the Sertoli cell intermediate filament cytoskeleton (Richburg and Boekelheide, 1996). The intermediate filament and celsr2 patterns following phthalate exposure are similar in both morphology and kinetics, suggesting a relationship between collapse of intermediate filaments and celsr2 redistribution.
Previous research described histopathological changes in the testis 3 h after phthalate exposure (Creasy et al., 1983; Dalgaard et al., 2001; Richburg and Boekelheide, 1996). In pubertal and postpubertal animal models of phthalate-induced testicular injury, the most prevalent alteration was germ cell sloughing into the seminiferous tubule lumen 6–12 h after phthalate exposure (Creasy et al., 1983). At high doses, changes in testicular gene expression are detectable at 2 h postexposure in both fetal (Liu et al., 2005) and pubertal models (our unpublished data). Because changes in gene expression often result from altered signaling processes (Karin, 1991), the observed changes in gene expression and histopathology are likely due to prior alterations in signal transduction pathways. We observed altered phosphorylation of celsr2 15 min after exposure in a model cell line and in vivo relocalization in rat testis within 2 h. The altered phosphorylation suggests that MEHP may produce an immediate change in testicular intracellular signaling prior to effects on gene expression and histopathological changes. Alterations in testicular signal transduction in response to MEHP have been documented for the transcription factor NF-
B (Rasoulpour and Boekelheide, 2005). The activation of NF-B is regulated, in part, by PKC (Catley et al., 2004; Ogasa et al., 2003), and our results suggest that MEHP may activate PKC. Therefore, MEHP-induced alterations in PKC activity may be responsible for some of the observed gene expression changes (Nishizuka, 1995) and raise the possibility that the activities of other kinases, such as CK1, and signaling molecules are altered by phthalate exposure.
The rapid onset of changes in celsr2 phosphorylation suggests that it is an early target during phthalate-induced testicular injury. Our research demonstrates that exposure to low concentrations of phthalate results in altered signaling which may have a negative impact on cellular homeostasis. Based upon the data presented here and in the literature, a hypothetical model of how phthalates induce adverse testicular effects was developed: (1) phthalates bind to their as yet unknown molecular target resulting in altered intracellular signaling, including phosphorylation, relocalization, and degradation of celsr2; (2) the altered signaling leads to changes in gene expression; and (3) the combined effects of phthalates on signaling and gene expression initiate changes in intercellular interactions and steroidogenesis, which produce the spectrum of phthalate-induced reproductive phenotypes.
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ACKNOWLEDGMENTS |
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We would like to thank Heather Bolstad for her help in making the celsr2-EGFP construct and Dr. Kevin Gaido, Dr. Dave Dorman, and the members of the Johnson laboratory for critically reading the manuscript and making helpful suggestions throughout the research. This work was supported by NIH grant R01 ES/HD11632.
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