1,25 dihydroxyvitamin D3 stimulates phospholipase C-gamma in rat colonocytes: role of c-Src in PLC-gamma activation.

Our laboratory has previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) rapidly stimulated polyphosphoinositide (PI) hydrolysis, raised intracellular Ca2+, and activated two Ca2+-dependent protein kinase C (PKC) isoforms, PKC-alpha and -betaII in the rat large intestine. We also showed that the direct addition of 1,25(OH)2D3 to isolated colonic membranes failed to stimulate PI hydrolysis, but required secosteroid treatment of intact colonocytes, suggesting the involvement of a soluble factor. Furthermore, this PI hydrolysis was restricted to the basal lateral plasma membrane of these cells. In the present studies, therefore, we examined whether polyphosphoinositide-phospholipase C-gamma (PI-PLC-gamma), a predominantly cytosolic isoform of PI-PLC, was involved in the hydrolysis of colonic membrane PI by 1,25(OH)2D3. This isoform has been shown to be activated and membrane-associated by tyrosine phosphorylation. We found that 1,25(OH)2D3 caused a significant increase in the biochemical activity, particulate association, and the tyrosine phosphorylation of PLC-gamma, specifically in the basal lateral membranes. This secosteroid also induced a twofold increase in the activity of Src, a proximate activator of PLC-gamma in other cells, with peaks at 1 and 9 min in association with Src tyrosine dephosphorylation. 1,25(OH)2D3 also increased the physical association of activated c-Src with PLC-gamma. In addition, Src isolated from colonocytes treated with 1,25(OH)2D3, demonstrated an increased ability to phosphorylate exogenous PLC-gamma in vitro. Inhibition of 1,25(OH)2D3-induced Src activation by PP1, a specific Src family protein tyrosine kinase inhibitor, blocked the ability of this secosteroid to stimulate the translocation and tyrosine phosphorylation of PLC-gamma in the basolateral membrane (BLM). Src activation was lost in D deficiency, and was reversibly restored with the in vivo repletion of 1,25(OH)2D3. These studies demonstrate for the first time that 1,25(OH)2D3 stimulates PLC-gamma as well as c-Src in rat colonocytes, and indicate that PLC-gamma is a direct substrate of secosteroid-activated c-Src in these cells.

Protein kinase C isoforms in the chemopreventive effects of a novel vitamin D3 analogue in rat colonic tumorigenesis.

BACKGROUND & AIMS: We recently showed that dietary supplementation with an analogue of 1alpha,25-dihydroxy-vitamin D3, 1alpha,25-dihydroxy-16-ene-23-yne-26,27 F6-vitamin D3 (RO24-5531), reduced the incidence of colonic tumors in rats treated with azoxymethane (AOM). The aim of this study was to determine whether alterations in specific isoforms of protein kinase C (PKC) are involved in this phenomenon. METHODS: Protein abundance and subcellular distribution of several PKC isoforms were examined and compared in AOM-induced tumors of rats fed control and RO24-5531-supplemented diets. RESULTS: In both AOM-induced colonic adenomas and carcinomas, a significant down-regulation of PKC-alpha, -delta, and -zeta and an up-regulation of PKC-beta11 were found compared with control colonocytes. Dietary RO24-5531 preserved the expression of PKC-zeta and increased the abundance of PKC-epsilon in carcinogen-induced adenomas. CONCLUSIONS: Because identical changes in specific isoforms of PKC were found in AOM-induced adenomas and carcinomas, these alterations may be involved in the early stage(s) of colonic malignant transformation. Moreover, the ability of RO24-5531 to block the changes in PKC-zeta induced by AOM, as well as to up-regulate PKC-epsilon, may underlie its ability to prevent adenomas from progressing to carcinomas.

Selective preservation of protein kinase C-zeta in the chemoprevention of azoxymethane-induced colonic tumors by piroxicam.

While nonsteroidal anti-inflammatory drugs have been shown to exert preventive effects against the development of colonic tumors in humans and in chemically-induced tumors in animal models, the mechanism(s) involved in this phenomenon is unclear. We have recently demonstrated that one such agent, piroxicam, when supplemented (75 ppm) in the diets of rats administered azoxymethane, reduced the incidence of rats bearing tumors. To date, the effects of piroxicam on protein kinase C, a family of serine/threonine kinases which may be intimately involved in the colonic malignant transformation process, have not been examined. It was, therefore, of interest to determine whether piroxicam altered the expression of one or more isoforms of this kinase in these tumors. The present studies demonstrate that dietary piroxicam selectively preserved the expression of protein kinase C-zeta in azoxymethane-induced tumors; suggesting that this is at least one mechanism involved in this agent's chemopreventive actions in this organ.

1,25-Dihydroxyvitamin D3 and 12-O-tetradecanoyl phorbol 13-acetate cause differential activation of Ca(2+)-dependent and Ca(2+)-independent isoforms of protein kinase C in rat colonocytes.

Considerable evidence that alterations in protein kinase C (PKC) are intimately involved in important physiologic and pathologic processes in many cells, including colonic epithelial cells, has accumulated. In this regard, phorbol esters, a class of potent PKC activators, have been found to induce a number of cellular events in normal or transformed colonocytes. In addition, our laboratory has demonstrated that the major active metabolite of vitamin D3, 1,25(OH)2D3, also rapidly (seconds-minutes) activated PKC and increased intracellular calcium in isolated rat colonocytes. These acute responses, however, were lost in vitamin D deficiency and partially restored with the in vivo repletion of 1,25(OH)2D3. The Ca(2+)-independent or novel isoforms of PKC expressed in the rat colon and the isoform-specific responses of PKC to acute treatment with phorbol esters or 1,25(OH)2D3 have not been previously characterized. Moreover, the effects of vitamin D status on PKC isoform expression, distribution, and response to agonists are also unknown. In the present experiments, in addition to PKC-alpha, rat colonocytes were found to express the novel isoforms delta, epsilon, and zeta by Western blotting using isoform-specific PKC antibodies. The tumor-promoting phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate, caused time- and concentration-dependent translocations of all these isoforms except PKC-zeta. In vitamin D deficiency, there were no alterations in colonic PKC isoform expression but significant changes in the subcellular distribution of PKC-alpha, -delta, and -zeta. Acute treatment of colonocytes from D-sufficient, but not D-deficient, rats with 1,25(OH)2D3 caused a rapid transient redistribution of only PKC-alpha from the soluble to the particulate fraction. The alterations in PKC isoform distribution and PKC-alpha responsiveness to 1,25(OH)2D3 in vitamin D deficiency were partially, but significantly, restored with 5-7 d in vivo repletion of this secosteroid. Both 12-O-tetradecanoyl phorbol 13-acetate and 1,25(OH)2D3 activated endogenous PKC, as assessed by inhibition of myristoylated alanine-rich C kinase substrate back-phosphorylation by exogenous PKC. These studies indicate that PKC-alpha, -delta, and/or -epsilon likely mediate important phorbol ester-stimulated events described in the rat colon. In contrast, PKC-alpha is implicated in the rapid (s-min) PKC-dependent events initiated by 1,25(OH)2D3 in rat colonocytes.

1,25-Dihydroxyvitamin D3 stimulates the phosphorylation of two acidic membrane proteins of 42,000 and 48,000 daltons in rat colonocytes: an effect modulated by vitamin D status.

Our laboratory has recently demonstrated that 1,25-dihydroxyvitamin D3(1,25(OH)2D3) rapidly stimulated membrane polyphosphoinositide breakdown and increased intracellular calcium, as well as activated protein kinase C (PKC) in vitamin D-sufficient rat colonocytes. These effects of 1,25(OH)2D3 were, however, lost in vitamin D-insufficient rats and restored by the in vivo repletion of 1,25(OH)2D3. In the present studies we have examined the ability of 1,25(OH)2D3 to stimulate the phosphorylation of colonic membrane proteins in intact D-sufficient cells. In addition, we investigated the effects of vitamin D status on the phosphorylation of these membrane proteins in broken cell preparations. These studies demonstrated that 1,25(OH)2D3 increased the phosphorylation of at least two colonic membrane proteins with apparent molecular weights of 42,000 (pp42) and 48,000 (pp48) in intact cells of vitamin D-sufficient rats. Moreover, in vitamin D-sufficient rats, treatment of colonocytes with 1,25(OH)2D3 or 12-O-tetradecanoyl phorbol 13-acetate (TPA), a known activator of PKC, significantly increased the phosphorylation of pp42 and pp48 in broken cell preparations. The kinetics of these phosphorylations in response to 1,25(OH)2D3 were both rapid and transient. In addition, PKC19-36, a specific PKC inhibitor, decreased the phosphorylation of pp42 and pp48, whereas okadaic acid (OA), a type 1 and 2A protein phosphatase inhibitor, further augmented their phosphorylation in response to 1,25(OH)2D3. The isoelectric points of pp42 and pp48 were 5.79 and 5.97, respectively, and both were predominantly phosphorylated on threonine residues. In contrast to our findings in colonocytes from vitamin D-sufficient animals, basal phosphorylation of pp42 and pp48 were increased in membranes prepared from vitamin D-insufficient rats. Moreover, these phosphorylations failed to change in response to 1,25(OH)2D3-treatment of colonocytes from vitamin D-insufficient rats. The basal phosphorylation of each of these proteins was restored to control levels, as was their ability to respond to the direct addition of 1,25(OH)2D3 following the in vivo repletion of vitamin D-insufficient rats with this secosteroid. In summary, we have identified two acidic membrane proteins from rat colonocytes that are phosphorylated in both intact and broken cell preparations in response to 1,25(OH)2D3 treatment, an event modulated by vitamin D status and mediated, at least in part, by PKC.

Guanylin activates rat colonic particulate guanylate cyclase.

The polypeptide guanylin is an endogenous activator of small intestinal guanylate cyclase. In rat, guanylin mRNA is found predominantly in intestinal tissues, with its highest abundance in the colon. To date, the effect of guanylin on rat colonic particulate guanylate cyclase, however, has not been examined. It was, therefore, of interest to determine whether the addition of guanylin to intact rat colonocytes, or directly to isolated crude colonic membranes, stimulated guanylate cyclase activity. These studies demonstrated that: 1) rat guanylin, in a concentration-dependent manner, rapidly (within min), but transiently, stimulated particulate guanylate cyclase activity when added to intact colonocytes; 2) guanylin also stimulated guanylate cyclase activity when added directly to isolated colonic membranes; and 3) this latter effect of guanylin on guanylate cyclase activity was increased by ATP or ADP and markedly accentuated by ATP gamma S. Taken together, these results demonstrate that guanylin rapidly stimulates rat colonic particulate guanylate cyclase activity and, moreover, that this effect can be modulated by adenine nucleotides.

1,25(OH)2 vitamin D3 activates PKC-alpha in Caco-2 cells: a mechanism to limit secosteroid-induced rise in [Ca2+]i.

Our laboratory recently reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly increases the breakdown of membrane phosphoinositides, raises intracellular calcium concentration ([Ca2+]i), and translocates protein kinase C (PKC) from the cytosolic to the particulate fraction of Caco-2 cells. In the present experiments, we found that Caco-2 cells contained predominantly the alpha- and zeta-isoforms of PKC, with minimally detectable amounts of PKC-beta and -epsilon by Western blotting. 1,25(OH)2D3 and the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) each caused time-dependent translocations of PKC-alpha, but not PKC-zeta. TPA treatment of these cells for 24 h induced a significant concentration-dependent downregulation of PKC-alpha, but not PKC-zeta. Since PKC inhibits phospholipase C-induced mobilization of Ca2+ in other cells, we examined the effects of staurosporine and H-7, PKC inhibitors, and TPA on 1,25(OH)2D3-stimulated increase in [Ca2+]i. As previously demonstrated by our laboratory, 1,25(OH)2D3 caused a biphasic increase in [Ca2+]i, with an initial elevation (transient phase) followed by a sustained increase (plateau phase). We previously demonstrated that the transient phase is mediated, at least in part, by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] stimulated by the secosteroid. Acute pretreatment with staurosporine or H-7 caused a significant stimulation, whereas acute TPA pretreatment caused a significant inhibition of the 1,25(OH)2D3-induced increase in the transient phase of [Ca2+]i. Preincubation of Caco-2 cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxy-methyl ester (BAPTA-AM) abolished both the rise in [Ca2+]i and the increase in particulate-associated PKC-alpha stimulated by 1,25(OH)2D3. Moreover, downregulation of PKC-alpha by chronic TPA treatment significantly augmented the transient phase of the 1,25(OH)2D3-stimulated rise in [Ca2+]i but had no effect on the 1,25(OH)2D3-induced change in Ins(1,4,5)P3 concentration. Furthermore, in these PKC-alpha downregulated cells staurosporine no longer increased the secosteroid-stimulated transient rise in [Ca2+]i. These results indicate that 1,25(OH)2D3, which increases [Ca2+]i and diacylglycerol, activates PKC-alpha, but not PKC-zeta. The alpha-isoform, in turn, limits the secosteroid-stimulated rise in [Ca2+]i, at a step distal to Ins(1,4,5)P3 accumulation in Caco-2 cells.

TPA causes divergent responses of Ca(2+)-dependent and Ca(2+)-independent isoforms of PKC in the nuclei of Caco-2 cells.

The present studies were undertaken to examine the expression of PKC isoforms within the nucleus of Caco-2 cells, a cell line widely used to investigate intestinal cell growth and differentiation, in order to begin to explore their roles in modulating gene expression. Purified nuclei were, therefore, prepared from Caco-2 cells and found to contain PKC-zeta, but not -alpha. The phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA) caused an acute redistribution of PKC-alpha to the nucleus, but did not change the distribution of PKC-zeta. Chronic treatment with TPA down-regulated total PKC-alpha, but not -zeta. Moreover, in contrast to acute TPA treatment, after chronic treatment, nuclear PKC-alpha was no longer detectable, whereas nuclear PKC-zeta was unchanged. These studies demonstrate for the first time the constitutive expression and divergent responses to TPA of the Ca(2+)-dependent and Ca(2+)-independent isoforms of PKC in the nuclei of Caco-2 cells and suggest that these specific isoforms may be involved in modulating gene expression.