1,25-Dihydroxyvitamin D3 selectively translocates PKCα to nuclei in ROS 17/2.8 cells

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Abstract

We have investigated protein kinase C (PKC) regulation by 1,25-(OH)2D3 in the rat osteosarcoma cell line ROS 17/2.8 since previous reports have implicated PKC in the 1,25-(OH)2D3-mediated regulation of osteocalcin gene expression (J. Biol. Chem. 267 (1992) 12562; Endocrinology 136 (1995) 5685). Here we report that 1,25-(OH)2D3 increased PKCα, but not PKCβI, ε or ζ, levels in the nuclear fraction in a time-dependent manner. Unlike PMA, 1,25-(OH)2D3 did not alter the association of any of the expressed PKC isoenzymes with the plasma membrane. Treatment with 20 nM 1,25-(OH)2D3 for 15 min, 30 min, 1 h and 24 h increased PKCα levels in the nuclear fraction by 2.3- to 2.6-fold. Nuclear PKCα expression was also increased with doses of 1,25-(OH)2D3 as low as a 0.05 nM. 1,25-(OH)2D3-mediated stabilization of osteocalcin mRNA (Arch. Biochem. Biophys. 332 (1996) 142) was inhibited with bisindolylmaleimide treatment, suggesting that PKCα may be involved in the 1,25-(OH)2D3-mediated regulation of osteocalcin gene expression.

Introduction

1,25-(OH)2D3, the active form of vitamin D, controls calcium and phosphorous homeostasis by acting through intracellular receptors and regulating gene expression in target tissues such as the intestine, kidney and bone (Darwish and DeLuca, 1993). While the actions of 1,25-(OH)2D3 in the intestine are well characterized, the mechanism by which 1,25-(OH)2D3 mediates mineral homeostasis in bone is still not well understood. Bone turnover is mainly regulated by osteoblasts and osteoclasts. While osteoclasts are involved in bone resorption, the osteoblasts main function is to synthesize new bone (Marks and Hermey, 1996). 1,25-(OH)2D3 has been shown to enhance osteoclastic activity, but receptors for 1,25-(OH)2D3 have not been identified in these bone-resorbing cells. It has been proposed that under the influence of 1,25-(OH)2D3, osteoblasts secrete a factor(s) that increases osteoclastic bone resorptive activity (McSheehy and Chambers, 1987). Osteocalcin, the major non-collagenous protein present in the extracellular matrix and exclusively produced by osteoblasts, has been proposed as a possible candidate (Lian et al., 1984, Glowacki and Lian, 1987).

The regulation of osteocalcin has been used as a model to study the actions of 1,25-(OH)2D3 in bone. 1,25-(OH)2D3 has been shown to regulate osteocalcin gene expression at the transcriptional level (Demay et al., 1989, Breen et al., 1994, Mosavin and Mellon, 1996). However, the molecular mechanism(s) by which 1,25-(OH)2D3 regulates the expression of the osteocalcin gene remains to be elucidated.

Several studies have implicated PKC as a component in the signaling pathway of 1,25-(OH)2D3 in a variety of cell lines (Simboli-Campbell et al., 1994, Berry et al., 1996, Bissonnette et al., 1994, Capiati et al., 1999, Simpson et al., 1998). Protein Kinase C is a family of serine/threonine kinases that consist of at least 12 isoenzymes. These isoforms differ in tissue expression, intracellular localization and in enzymological properties (Dekker and Parker, 1994, Nishizuka, 1995). Based on their structure and mode of activation, they are divided into three groups. The classical or conventional isoforms (α, βI, βII and γ) are activated by Ca2+, diacylglycerol (DAG), phorbol esters, phosphatidylserine (PS) and cis-unsaturated fatty acids. The novel isoforms (δ, ε, η(L), θ and μ) are regulated by DAG, phorbol esters and PS, but not by Ca2+. The last group, the atypical isoforms (ζ, ι and λ) are activated by PS, but not by Ca2+, DAG or phorbol esters. These enzymes mediate a variety of cellular responses, which are triggered by a vast array of agonists such as neurotransmitters, growth factors, hormones and drugs. Besides PKC's role in signal transduction events, emerging evidence suggest a role of PKC in ‘long term’ cellular functions such as proliferation and differentiation (Solomon et al., 1991, Brooks et al., 1993, Dekker and Parker, 1994, Gamard et al., 1994, Nishizuka, 1995, Simpson et al., 1998, Capiati et al., 1999, Capiati et al., 2000).

Evidence for the functional involvement of PKC in the actions of 1,25-(OH)2D3 in bone with respect to osteocalcin regulation has been reported. Van Leeuwen et al., reported that treatment of ROS 17/2.8 cells with the PKC inhibitors 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG) and sphingosine inhibit 1,25-(OH)2D3-stimulated osteocalcin synthesis (van Leeuwen et al., 1992). In another study, the PKC inhibitor staurosporine was shown to inhibit the 1,25-(OH)2D3-mediated induction of osteocalcin gene transcription (Desai et al., 1995).

An important criteria for establishing the relevance of PKC inhibitor data, is to demonstrate PKC isoform expression and regulation by 1,25-(OH)2D3. It is known that PKC isoenzymes are present predominantly in the cytoplasm in an inactive state and upon activation they translocate to other cellular compartments. 1,25-(OH)2D3 has been shown to regulate PKC activity in several cell lines. In Madin Darby bovine kidney (MDBK) cells, treatment with 1,25-(OH)2D3 increased plasma membrane association of PKCα and induced translocation of PKCβ to the nuclei (Simboli-Campbell et al., 1994). In human promyelocytic leukemia NB4 cells, 1,25-(OH)2D3 induced translocation of PKCδ to the nuclei, and PKCα to the nuclei and to a particulate fraction (Berry et al., 1996). In human colon cancer-derived Caco-2 cells, 1,25-(OH)2D3 activates PKCα and this isoform in turn limits the 1,25-(OH)2D3-stimulated rise in intracellular Ca2+ (Bissonnette et al., 1994). In chick myoblasts the early 1,25-(OH)2D3-mediated stimulation of myoblast proliferation correlated with an increase in PKCα expression whereas decreased PKCα levels are observed during the subsequent 1,25-(OH)2D3-induction of myoblast differentiation (Capiati et al., 1999). PKC (specifically the α isoform) has also been implicated in the 1,25-(OH)2D3 regulation of intracellular Ca2+ during development of skeletal muscle cells (Capiati et al., 2000). However, to our knowledge, regulation of PKC isoforms by 1,25-(OH)2D3 in bone cells has not been reported.

The present study was designated to investigate the effect of 1,25-(OH)2D3 on PKC translocation/activation to the nuclei and plasma membrane in the rat osteosarcoma cell line ROS 17/2.8. We first determined the presence of detectable isoforms, which included PKCα, βI, ζ and ε in ROS 17/2.8 cells by western blotting. Western blotting also was employed to characterize the regulation of these PKC isoforms by 1,25-(OH)2D3. We determined that 1,25-(OH)2D3 increased PKCα, but not PKCζ, βI and ε, levels in the nuclear fraction, in a time-dependent manner. Whereas, 1,25-(OH)2D3 did not alter the levels of any of the expressed PKC isoforms in the plasma membrane. The increase in PKCα levels in the nuclear fraction was observed as early as 15 min after treatment with 1,25-(OH)2D3. Epi-fluorescence and confocal microscopy corroborated the 1,25-(OH)2D3-mediated increase in PKCα levels in the nuclei that was observed by immunoblotting. To our knowledge, this is the first study that reports regulation of PKCα by 1,25-(OH)2D3 in bone cells.

Since PKC has been implicated in 1,25-(OH)2D3-mediated gene expression, we tested whether inhibitors of PKC would affect 1,25-(OH)2D3-induced osteocalcin mRNA expression. Calphostin C and bisindolylmaleimide reduced the steady state levels of osteocalcin mRNA accumulated in ROS 17/2.8 cells treated with 1,25-(OH)2D3. Moreover, previously published results demonstrated that 1,25-(OH)2D3 regulates osteocalcin expression primarily at the posttranscriptional level (Mosavin and Mellon, 1996). Inhibition of PKC by the PKC potent inhibitor bisindolylmaleimide blocked the 1,25-(OH)2D3-mediated stabilization of osteocalcin mRNA expression. This suggests that PKCα may be involved in the l,25-(OH)2D3-mediated regulation of osteocalcin mRNA since 1,25-(OH)2D3 regulated PKCα but not any of the other expressed PKC isoforms in this cell line.

Section snippets

Cell culture

ROS 17/2.8 cells were maintained in Ham's F-12 (1:5) culture medium supplemented with 5% (vol/vol) defined fetal bovine serum (Hyclone, Utah), penicillin (100 U/ml), and streptomycin (0.1 mg/ml). Cells were propagated continuously at 37 °C under a humidified 5% CO2: 95% air mixture. For the experiments, cells were plated at a density of 1.0×104 cells/cm2 on either 55 cm2 petri dishes (for immunoblotting and northern blotting) or 15 mm glass coverslips (for immunofluorescence), and allowed to

Expression of PKC isoenzymes in ROS 17/2.8 cells

An important criteria to establish the relevance of PKC to the 1,25-(OH)2D3 intracellular signaling pathway is to show the expression and regulation of PKC in bone cells. First, ROS 17/2.8 cells were examined for PKC isoform expression. As shown in Fig. 1, PKCα, βI, ζ and ε are present in ROS 17/2.8 cells, while PKCβII and PKCγ were not detected. It is apparent from the results of multiple experiments that PKCα and PKCζ are the most abundant isoforms in this cell line.

1,25-(OH)2D3 has been

Discussion

We are interested in determining the signaling pathway(s) by which 1,25-(OH)2D3 regulates osteocalcin gene expression. Previous studies have implicated PKC as a component of the signaling pathway of 1,25-(OH)2D3 in bone, particularly in the regulation of the osteocalcin gene (van Leeuwen et al., 1992, Desai et al., 1995). This led us to study PKC isoform expression and regulation by 1,25-(OH)2D3 in ROS 17/2.8 cells. We determined that 1,25-(OH)2D3 specifically increased PKCα, but not PKCζ, βI

Acknowledgements

We thank Dr. Shyh-Min Huang, Department of Biomolecular Chemistry, University of Wisconsin–Madison, for expert technical assistance with immunoblotting; Dr. Chris M. Erickson, Julian F. Hillyer and Paul A. Sims, Department of Animal Health and Biomedical Sciences, University of Wisconsin–Madison, for their expert technical assistance with fluorescence microscopy; Allison Doyle, Division of Pharmaceutical Sciences, University of Wisconsin–Madison, for assistance with the immunofluorescence

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