Beth Scaglione-Sewell

1,25-Dihydroxyvitamin D3 Stimulates Activator Protein-1-dependent Caco-2 Cell Differentiation

1,25-Dihydroxyvitamin D3(1,25(OH)2D3) is a potential chemopreventive agent for human colon cancer. We have reported that 1,25(OH)2D3 specifically activated protein kinase C-α (PKC-α) and also caused a reduction in proliferation while increasing apoptosis and differentiation in CaCo-2 cells, a cell line derived from a human colon cancer. The mechanisms by which this secosteroid influences these important cellular processes, however, remain unclear. The transcription factor, activator protein-1 (AP-1), regulates many genes involved in these processes. Therefore, we asked whether 1,25(OH)2D3 activated AP-1 in CaCo-2 cells and, if so, by what mechanisms? 1,25(OH)2D3 caused a time-dependent increase in AP-1 DNA binding activity and significantly enhanced the protein and mRNA abundance of c-Jun, a component of AP-1. 1,25(OH)2D3 also induced a rapid and transient activation of ERK2 (where ERK is extracellular signal-regulated kinase) and a more persistent activation of JNK1 (where JNK Jun N-terminal kinase). Transfection experiments revealed that 1,25(OH)2D3 also increased AP-1 gene-transactivating activity. This AP-1 activation was completely blocked by PD 098059, a specific mitogen-activated protein kinase/ERK kinase inhibitor, as well as by a dominant negative JNK or a dominant negative Jun, indicating that the AP-1 activation induced by 1,25(OH)2D3 was mediated by ERK and JNK. Using a specific inhibitor of the Ca2+-dependent PKC isoforms, Gö6976, and CaCo-2 cells stably transfected with antisense PKC-α cDNA, demonstrated that PKC-α mediated the AP-1 activation induced by this secosteroid. Inhibition of JNK activation or c-Jun protein expression significantly reduced 1,25(OH)2D3-induced alkaline phosphatase activity, a marker of CaCo-2 cell differentiation, in secosteroid-treated cells. Taken together, the present study demonstrated that 1,25(OH)2D3 stimulated AP-1 activation in CaCo-2 cells by a PKC-α- and JNK-dependent mechanism leading to increases in cellular differentiation.

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.

Rapid effects of 1,25(OH)2 vitamin D3 on signal transduction systems in colonic cells

Previous work from our laboratory demonstrated that 1,25(OH)2D3 rapidly stimulated hydrolysis of membrane polyphosphoinositides (PI) in rat colonocytes and in Caco-2 cells, generating the second messengers DAG and IP3. [Ca2+]i subsequently increased due to IP3-mediated release of intracellular Ca2+ stores, and to Ca2+ influx through a receptor-mediated Ca channel. Studies examining purified antipodal plasma membranes and experiments using Caco-2 cell monolayers found that 1,25(OH)2D3 influenced PI turnover only in the basolateral (BLM) and not brush border (BBM) membranes. Vitamin D analogues with poor affinity for the vitamin D receptor were found to effectively stimulate PI turnover, suggesting the presence of a unique vitamin D receptor in the BLM. Studies from our laboratory have demonstrated saturable, reversible binding of 1,25(OH)2 D3 to colonocyte BLM. Recently, we found that 1,25(OH)2D3 activated the tyrosine kinase c-src in colonocyte BLM by a heterotrimeric guanine nucleotide binding protein (G-protein)-dependent mechanism, with subsequent phosphorylation, translocation to the BLM, and activation of PI-specific phospholipase C gamma. Due to the rise in [Ca2+]i and DAG, two isoforms of protein kinase C (PKCalpha and PKCbeta2), but not other isoforms were activated by 1,25(OH)2D3 in rat colonocytes. Recent studies demonstrated that the seco-steroid translocated the beta2 isoform to the BLM, but not the BBM. In contrast, the alpha isoform did not translocate to either antipodal plasma membrane, but modulated IP3-mediated Ca2+ release from the endoplasmic reticulum. Preliminary studies have shown that 1,25(OH)2D3 also activated phosphatidylcholine phospholipase D (PLD) in Caco-2 cells, generating phosphatidic acid and contributing to the sustained rise in DAG. PLD stimulation occurred by both PKC-dependent and -independent mechanisms. Inhibitors of G-proteins, c-src, and PKC blunted the seco-steroid-mediated activation of PLD. Cells stably transfected with sense PKCalpha showed increased 1,25(OH)2D3-stimulated PLD activation, whereas transfectants with antisense PKCalpha had an attenuated response. In addition, 1,25(OH)2D3 also regulated PLD by activating the monomeric G-protein rho A by a mechanism independent of the G-protein/ c-src/PKC pathway.

1,25-Dihydroxyvitamin D3 but not TPA activates PLD in Caco-2 cells via pp60c-src and RhoA

In the accompanying paper [Khare et al., Am. J. Physiol. 276 ( Gastrointest. Liver Physiol. 39): G993-G1004, 1999], activation of protein kinase C-α (PKC-α) was shown to be involved in the stimulation of phospholipase D (PLD) by 1,25-dihydroxyvitamin D3[1,25(OH)2D3] and 12- O-tetradecanoylphorbol 13-acetate (TPA) in Caco-2 cells. Monomeric or heterotrimeric G proteins, as well as pp60c- src have been implicated in PLD activation. We therefore determined whether these signal transduction elements were involved in PLD stimulation by 1,25(OH)2D3or TPA. Treatment with C3 transferase, which inhibits members of the Rho family of monomeric G proteins, markedly diminished the ability of 1,25(OH)2D3, but not TPA, to stimulate PLD. Brefeldin A, an inhibitor of ADP-ribosylation factor proteins, did not, however, significantly reduce the stimulation of PLD by either of these agents. Moreover, 1,25(OH)2D3, but not TPA, activated pp60c- src and treatment with PP1, a specific inhibitor of the pp60c- src family, blocked the ability of 1,25(OH)2D3to activate PLD. Pretreatment of cells with pertussis toxin (PTx) markedly reduced the stimulation of PLD by either agonist. PTx, moreover, inhibited the stimulation of pp60c- src and PKC-α by 1,25(OH)2D3. PTx did not, however, block the membrane translocation of RhoA induced by 1,25(OH)2D3or inhibit the stimulation of PKC-α by TPA. These findings, taken together with those of the accompanying paper, indicate that although 1,25(OH)2D3and TPA each activate PLD in Caco-2 cells in part via PKC-α, their stimulation of PLD differs in a number of important aspects, including the requirement for pp60c- src and RhoA in the activation of PLD by 1,25(OH)2D3, but not TPA. Moreover, the requirement for different signal transduction elements by 1,25(OH)2D3and TPA to induce the stimulation of PLD may potentially underlie differences in the physiological effects of these agents in Caco-2 cells.

1,25-Dihydroxyvitamin D3 and TPA activate phospholipase D in Caco-2 cells: role of PKC-α

1,25-Dihydroxyvitamin D3[1,25(OH)2D3] and 12- O-tetradecanoylphorbol 13-acetate (TPA) both activated phospholipase D (PLD) in Caco-2 cells. GF-109203x, an inhibitor of protein kinase C (PKC) isoforms, inhibited this activation by both of these agonists. 1,25(OH)2D3activated PKC-α, but not PKC-β1, -βII, -δ, or -ζ, whereas TPA activated PKC-α, -β1, and -δ. Chronic treatment with TPA (1 μM, 24 h) significantly reduced the expression of PKC-α, -βI, and -δ and markedly reduced the ability of 1,25(OH)2D3or TPA to acutely stimulate PLD. Removal of Ca2+ from the medium, as well as preincubation of cells with Gö-6976, an inhibitor of Ca2+-dependent PKC isoforms, significantly reduced the stimulation of PLD by 1,25(OH)2D3or TPA. Treatment with 12-deoxyphorbol-13-phenylacetate-20-acetate, which specifically activates PKC-βI and -βII, however, failed to stimulate PLD. In addition, the activation of PLD by 1,25(OH)2D3or TPA was markedly reduced or accentuated in stably transfected cells with inhibited or amplified PKC-α expression, respectively. Taken together, these observations indicate that PKC-α is intimately involved in the stimulation of PLD in Caco-2 cells by 1,25(OH)2D3or TPA.

Alterations in protein kinase C-bII in CaCo-2 cells result in changes in both cell growth and differentiation

Protein kinase C (PKC)consists of a gene family of serine/threonine kinases which regulate numerous events including cellular proliferation and differentiation. Studies in human and experimental models of colon cancer suggest that alterations in the expression of specific isoforms of PKC are involved in colonic malignant transformation. Previously, our laboratory has shown that PKC/3II protein expression was altered in azoxymethane-induced rat colonic tumors. The specific aim of this study was to characterize the functional consequences of alterations in PKC/311 in CaCo-2 cells, a colonic adenocarcinoma cell line. Methods: Full length human PKC/3II cDNA, and a kinase-dead isoform mutated in the catalytic domain, were cloned into MRE, a Zn2+ inducible metallothionine expression vector. Mutant and wild type sequences were confirmed by direct sequencing. CaCo-2 cells were transfected, selected with G418 and polyclonal populations were expanded . Cell Iysates were analyzed by western blotting for isoform specific PKC expression. Following immunoprecipitation with PKC/3II specific antibodies, kinase activity was measured using acetylated myelin basic protein substrate. Proliferation was analyzed by cell counting. Differentiation was assessed by alkaline phosphatase . Results: In the presence of 175 p,M Zn2+, CaCo-2 cells transfected with both wild type PKC/3II and kinase-dead PKC/3II exhibited 3-4 -fold increases in the expression of the isoform compared to empty vector (EV) transfectants as assessed by Western blotting. Transfection with kinase-dead PKC/3II significantly inhibited (p :s 0.05) kinase activity by nearly 70%. Importantly, the mutant and wild type clones exhibited no significant changes in the expression of non-targeted PKC isoforms, including PKCa,/3I,/l or ~. While the wild type PKC/3II transfected CaCo-2 cells did not differ in proliferation compared to the EV cells, kinase-dead transfectants showed more than a 30% increase (p :s 0.05) in cell growth compared to EV control s. In addition, by day I I post-plating, these kinase-dead PKC/3II transfected cells demonstrated a 25% decrease in alkaline phosphatase activity compared to EV controls. In conclusion, we have shown that Caco-2 cells, transfected with a kinase-dead PKC/3II cDNA and possessing decreased kinase activity, demonstrate enhanced proliferation and inhibited differentiation. These studies suggest that PKC/3II may be involved in the regulation of these important cellular processes in human colon cancer cells.