Activation of Protein Kinase-C Inhibits Vitamin D Receptor Gene Expression

Aruna V. Krishnan and David Feldman

The abundance of 1,25-dihydroxyvitamin D3 receptors (VDR) in cultured cells has been shown to vary in direct relation to the rate of cell proliferation. This study examines the question of whether the growthfactor mediated up-regulation of VDR is due to direct modulation of VDR gene expression or is secondary to the stimulation of cell cycle events. Mitogenic agents, such as basic fibroblast growth factor and phorbol esters, were found to cause significant decreases in VDR abundance, while substantially stimulating proliferation of NIH-3T3 cells. Potent phorbol esters, such as phorbol myristate acetate (PMA) and phorbol-12,13-dibutyrate, whose biological actions have been shown to be mediated through the activation of protein kinase-C, down-regulated VDR in a time- and dose-dependent manner. An inactive phorbol ester, 4a-phorbol-12,13-didecanoate, which does not activate protein kinase-C, did not alter VDR levels. Desensitization of protein kinase-C by prolonged exposure of cells to phorbol esters eliminated the PMA-mediated down-regulation of VDR. Staurosporine, an inhibitor of protein kinase-C, blocked the actions of PMA. Oleoyl acetyl glycerol, a synthetic diacyl glycerol, and A23187, a calcium ionophore, were both able to suppress VDR abundance alone and were additive in combination. The results suggest that activation of the protein kinaseC pathway and elevation of intracellular Ca2+ lead to significant down-regulation of VDR. The inhibitory effect of PMA appears to be exerted at the level of VDR mRNA expression. Northern blot analysis revealed significant decreases in steady state levels of VDR mRNA species that qualitatively corresponded to the decrease in VDR protein concentration seen on a Western blot. The present study also shows that the protein kinase-C-mediated inhibition of VDR gene expression could override the stimulation of VDR levels brought about by other mitogenic agents, such as serum. The findings allow us to uncouple the rate of cell proliferation and regulation of VDR abundance that have previously been shown to be closely associated with each other and

indicate that mitogens acting via specific transmembrane signaling pathways directly and differentially modulate VDR gene expression. (Molecular Endocrinology 5: 605-612, 1991)

INTRODUCTION

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the active form of vitamin D, binds to a high affinity vitamin D receptor (VDR) and acts in target cells in a manner analogous to the steroid hormones (1). In addition to its classical calciotropic actions (2), an expanded role for 1,25-(OH)2D3 has recently been recognized in the regulation of cell proliferation and differentiation (3). A number of studies have shown that 1,25-(OH)2D3 differentiates and slows the growth of several cell types, including promyelocytic leukemia (HL-60) cells (4, 5), malignant melanoma cells (6), macrophages (7), keratinocytes (8), bone (9), and colon cancer cells (10). Of particular interest is the fact that VDR levels are regulated in close association with the rate of cell proliferation in a number of cultured cells (11-13). VDR levels are high in log phase cells when the rate of proliferation is also high and fall to low levels as cells reach confluence and become quiescent (11,13). In NIH-3T3 mouse fibroblasts, mitogens, such as serum, epidermal growth factor (EGF), insulin-like growth factor-l (IGF-I), and high concentrations of insulin (presumably acting through the IGF-I receptor), have been shown to stimulate cell proliferation as well as cause an associated increase in the levels of VDR protein and mRNA (13). Further studies on growth factor regulation of VDR, to be presented in this paper, show that it is possible to uncouple the regulation of VDR gene expression from that of the cell proliferation rate. Some mitogens, such as basic fibroblast growth factor (b-FGF), were found to have the ability to suppress VDR levels while stimulating proliferation of NIH-3T3 mouse fibroblasts. Since b-FGF is known to activate Ca2+/phospholipid-dependent protein kinase-C (14,15), experiments were carried out to test other activators of protein kinase-C. Tumorpromoting phorbol esters were also found to act as regulators of VDR. Data will be presented to show that

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down-regulation of VDR by these mitogens is a protein kinase-C mediated event. Moreover, b-FGF and phorbol esters can abolish the stimulation of VDR abundance in 3T3 fibroblasts caused by other mitogenic agents, such as serum. The fall in VDR levels is mediated by decreases in the steady state levels of VDR mRNA.

RESULTS

When confluent quiescent cultures of 3T3 cells were stimulated with serum, there was a substantial (6-fold) increase in the rate of cell proliferation, as measured by [3H]thymidine incorporation, a result consistent with our previous studies (13). This rise was associated with a 5-fold increase in VDR abundance, as measured by [3H] 1,25-(OH)2D3 binding (Fig. 1). Other mitogens, such as EGF and IGF-I, have also been shown to stimulate both proliferation rate and VDR levels in these cells (13). However, when the cells were treated with b-FGF or PMA in a fresh basal medium without serum [Dulbecco's Modified Eagle's Medium (DMEM) plus 0.1% BSA],

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Effect of Phorbol Esters on VDR In experiments designed to more closely examine the effects of phorbol esters, quiescent 3T3 cells were stimulated for 14 h with 10% serum before study to ensure high levels of VDR at the start of the experiment. The time course of PMA action on the cells is shown in Fig. 2. At the time of PMA addition (time zero), serumstimulated cells had a mean VDR level of 50 ± 2.6 fmol/ mg (100%). At this point one set of cells received the vehicle [dimethylsulfoxide (DMSO)] and served as control, while a second set of cells was treated with PMA

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