Trevor W. Lissoos$, David W. A. Beno, and Bernard H. Davis$. From the ..... thank Dr. U. Rapp (National Cancer Institute) and Dr. V. Sukhatme. (University of ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Val. 268, No.33, Issue of November 25, pp. 25132”25138,1993 Printed in U.S. A.
Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
1,25-Dihydroxyvitamin D3 Activates Raf Kinase and RafPerinuclear Translocation via a Protein Kinase C-DependentPathway* (Received for publication, May 22, 1993)
Trevor W. Lissoos$, David W. A. Beno, and Bernard H. Davis$ From the Gastroenterology Section, Department of Medicine, University of Chicago, Chicago, Illinois 60637
1,26-Dihydroxyvitamin D3’s (D3) potential mitogenic mechanism ofaction was pursued in cultured rat hepatic Ito cells, a fibrogenic effector cell which proliferates in vivo during liver injury and fibrogenesis. D3 stimulated Ito cell DNA synthesis and potentiated platelet-derived growth factor-induced mitogenesis. D3’s enhancement of [‘Hlthymidine incorporation was associated with nuclear Egr expression. ,Recentstudies have causally linked the activated proto-oncogene cRaf with downstream Egr induction. The serine-threonine kinase Raf protein is phosphorylation-activated by a large array of agonists including plasma membrane and cytoplasmic tyrosine kinases but has not previously been associated with the steroid superfamily of mediators. To consider potential prenuclear acute pathways of D3-induced stimulation, the activation of Raf was examined following D3 exposure. D3 induced Rafactivation as assessed via (a)enhanced Raf phosphorylation following in vivo’‘P labeling, ( b ) enhanced kinase function utilizing exogenous histone 1 protein as substrate, and ( c ) the shift in Raf physical localization changing from a diffuse cytoplasmic distribution to a perinuclear domain. A similar activation of Raf kinase was found in 3T3 cells exposed to D3 with enhanced histone phosphorylation detectable within 1 min following stimulation. The proximal cascade leading to Raf kinase activation may involve a protein kinase C-dependent pathway, sincevitamin Dstimulated kinase activity was severely attenuated by previous phorbol ester treatment for 20 h or staurosporine pretreatment.
nuclear vitamin D receptor is generally regarded as playing a central role (1). This latterreceptor is a member ofthe steroid superfamily of receptors and theprenuclear eventsleading to receptor interactions have not been well explored (1).However, recent studies involving intact isolated colonic epithelia as well as CACO-2 intestinal cell cultures suggest that D3 can cause acute changes in intracellular calcium and inositol 1,4,5triphosphate formation (2, 3). The dominant mechanism(s) of action for these nongenomic effects are unknown, yet they may be distinct from the better studied genomic effects, since preliminary work has also found that vitamin D analogues with markedly different affinities for the nuclear vitamin D receptor have similar effects on intracellular calcium (4). As inositol 1,4,5-triphosphate formation is often associated with protein kinase C (PKC) metabolism, it is possible that PKC could play a key role in these nongenomic effects (5). Furthermore, as no plasma membrane receptor for D3 has been identified, it is possible that PKC directly or indirectly could play this role. Using the HL-60 promyelocytic leukemia cell line, previous studies have in fact shown that D3 can upregulate the rate of transcription of PKC as well as the endogenous phosphorylation of PKC substrates without exogenous activation or endogenous diacylglycerol formation (6). In addition, D3 modulation of HL-60 differentiation and c-myc transcription is blocked byinhibitors of PKC (7). These studies were performed over a 4-24-h time course, and therefore, the acute effects of D3 and the mechanistic role of PKC in this pathway are unknown (6,7). Recent preliminary work, however, in CACO-2 cells has found that phorbol 12-myristate 13-acetate (PMA) induced down modulation of PKC-a enhanced D3’s acute (in minutes)induction of cytosolic calcium, suggesting a role for PKC in the negative feedback regulation of D3-induced calcium rises (5). 1,25-Dihydroxyvitamin D3 (D3)’ has been identified as a These mechanistic studies have largely been done in maligmajor differentiation agent in a variety of cell types (1).Its nant derived cell lines. However, D3’s physiologic rolerelates biphasic effects with regards to cell proliferation (i.e. both to its traditional in vivo kidney, intestine, and bone targets stimulatory and inhibitory) as well as its variable effects on as well nuclear D3 receptor expression in heart, brain, and gene transcription are incompletely understood, although the breast tissue (1).In addition, recent studies have found D3 effects for circulating monocytes as well as freshly * This work was supported in part by the Liver Research Fund, mitogenic isolated aortic tissue and cultured vascular smooth muscle University of Chicago and National Institutes of HealthGrants DK40223, DK 42086, and DK 07074-18. The costs of publication of cells (8, 9). To extend the potential range of D3 targets as this article were defrayed in part by the payment of page charges. well as to explore prenuclear D3 pathways, the current study This article must therefore be hereby marked “advertisement” in examined D3’s effects on rat hepatic sinusoidal Ito cells. This accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. cell type undergoes activation and proliferation during liver $ Current address: Gastroenterology Division, Dept. of Medicine, injury and fibrogenesis in vivo and is a major source of the Washington University School of Medicine, 660 S. Euclid, P. 0. BOX extracellular collagen matrix which accumulates during the 8124, St. Louis, MO 63110. T o whom correspondence should be addressed: Gastroenterology cirrhotic process during the formation of constrictive nodules Section, Dept. of Medicine, MC 4076, 5841 S. Maryland Ave., Uni- (10). The factors that mediate and perpetuate thispathologic versity of Chicago, Chicago, IL 60637. Tel.: 312-702-1467; Fax: 312- activation are poorly understood, but they remain a central 702-2182. issue in ultimately understanding and regulating the fibroThe abbreviations used are: D3, vitamin Da; PKC, protein kinase C; PMA, phorbol 12-myristate13-acetate;PDGF, platelet-derived genic process. In the current work, D3 was found to be mitogenic for Ito growth factor; FCS, fetal calf serum; PBS, phosphate-buffered saline; cells and to induce nuclear Egr expression. To examine poBSA, bovine serum albumin.
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Vitamin D3 Activates Raf Kinase
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exogenous substrate for phosphorylation. The reaction was terminated by adding 4 X solubilization buffer, heated to 95 "C X 3 min, and processed as described above. Eluates were loaded on a 14% SDSgel and the gel containing the resolved proteins was subsequently fixed, stained, dried, and exposed to Kodak XAR-5 film. Relative kinase activity was determined by scanning the histone-labeled band with a laser densitometer. Representative kinase assays are shown and were repeated in two to threeseparate experiments. TOsuppress protein kinase C activity, the cells were either pretreated with PMA (800 nm) X 20 h or the PKC inhibitor, staurosporine (20 nm) X 1 h. When the acute effects of PMA were compared, Ito cells were exposed to PMA (100 nm) X 15 min. The Raf cytoplasm to perinuclear translocation was assessed during Ito cell culture in response to D3 as described previously for It0 cell serum or PDGF stimulation (17). This enabled an assessment of the capacity of the activated Raf protein to physically shift its localization which might be important in the further downstream MATERIALS ANDMETHODS transmission of the mitogenic cascade. Ito cells were cultured in 24Chernicals-1,25-dihydroxyvitamin D3 (D3)(Steroids Ltd., Chi- well plates and made quiescent with overnight culture in 0.4% FCS cago) used in the experiments was stored as a stock solution in 100% as described above and thenexposed to D3 for 30 min. The cells were ethanol under argon in a brown bottle a t 4 "C until use. For experi- then washed with iced PBS X 2, fixed in methanol X 10 min, washed ments, D3 was freshly diluted in tissue culture media. Control cultures sequentially in PBS followed by PBS + 1%bovine serum albumin treated with equivalent volumes of ethanol vehicle (final concentra- (PBS/BSA), and blocked in normal horse serum (1:lO dilution in tion
