Regulation of rat ileal NHE3 by 1,25(OH)2-vitamin D3.

We have previously demonstrated a modulation of Na+/H+ exchange (NHE) activity by vitamin D3 in the rat ileum and Caco-2 cells. However, the molecular mechanism(s) of action of vitamin D3 on NHE are still not understood. The current studies were undertaken to understand the regulation of individual NHE isoforms on mRNA levels in two distinct models of vitamin D3 deficiency. Acute D3 deficiency was induced secondary to streptozotocin-induced diabetes mellitus, while chronic D3 deficiency was induced by feeding a D3-deficient diet in an environment devoid of fluorescent light. Vitamin D3 deficiency in both models increased the initial rates of rat ileal brush-border membrane (BBM) Na+/H+ exchange by 2.5-fold compared to D-repleted controls. In parallel to the increased exchanger activity, NHE3 mRNA abundance was increased about twofold in both acute and chronic D deficiency compared to control. There was no change in NHE1 or NHE2 abundance in vitamin D3-deficient rat ileum. These findings indicate that vitamin D3 regulates Na+/H+ exchange activity in rat ileum by influencing the mRNA levels of NHE3, the predominant luminal membrane isoform involved in vectorial Na+ transport.

Differential regulation of the expression of Na(+)/H(+) exchanger isoform NHE3 by PKC-alpha in Caco-2 cells.

Na(+)/H(+) exchange (NHE) activity has been shown to be regulated by various external signals and protein kinases in many tissues and cell types. A family of six NHE isoforms has been identified. Three isoforms, NHE1, NHE2, and NHE3, have been shown to be expressed in the human intestine. The present studies were designed to study regulation of these human NHE isoforms by the alpha-isoform of protein kinase C (PKC) in the Caco-2 cell line. The mRNA levels of the NHE isoforms in Caco-2 cells were initially measured by a semiquantitative RT-PCR technique in response to PKC downregulation by long-term exposure to 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h. PKC downregulation resulted in an approximately 60% increase in the mRNA level for NHE3, but not for NHE1 or NHE2. Utilizing dichlorobenzimidazole riboside, an agent to block the synthesis of new mRNA, we demonstrated that the increase in the NHE3 mRNA in response to downregulation of PKC was predominantly due to an increase in the rate of transcription, rather than a decrease in the NHE3 mRNA stability. Consistent with the mRNA results, our data showed that amiloride-sensitive (22)Na(+) uptake was increased after incubation of Caco-2 cells with 1 microM TPA for 24 h. To elucidate the role of PKC-alpha, an isoform downregulated by TPA, the relative abundance of NHE isoform mRNA levels and the apical NHE activity were assessed in Caco-2 cells over- and underexpressing PKC-alpha. Our results demonstrated that NHE3, but not NHE1 or NHE2, mRNA was downregulated by PKC-alpha and that apical NHE activity was higher in cells underexpressing PKC-alpha and lower in cells overexpressing PKC-alpha than in control cells. In conclusion, these data demonstrate a differential regulation of NHE3, but not NHE2 or NHE1, expression by PKC in Caco-2 cells, and this regulation appears to be predominantly due to PKC-alpha.

PKC-delta inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases apoptosis in CaCo-2 cells.

BACKGROUND & AIMS: Previous studies showed decreased protein kinase C (PKC)-delta expression in azoxymethane-induced rat and sporadic human colonic tumors. To elucidate the role of PKC-delta on the neoplastic phenotype of human colon cancer cells, we established stable transfectants of this isoenzyme in CaCo-2 cells. METHODS: Human PKC-delta complementary DNA was subcloned into 2 distinct metallothionein-regulated expression vectors. Polyclonal populations of PKC-delta transfectants were characterized by Western blotting. PKC-delta activity was measured in situ using a PKC-delta-specific substrate. Proliferation was determined by Coulter counter, and cell cycle distribution was analyzed by flow cytometry. In vitro transformation was assessed by growth in soft agar and differentiation by changes in alkaline phosphatase and sucrase isomaltase. Apoptosis was evaluated by 4',6-diamidino-2-phenylindole dihydrochloride and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining. RESULTS: In the presence of Zn(2+), PKC-delta transfectants expressed a 4-fold increase in the protein and a 2-fold increase in activity of PKC-delta. PKC-delta transfectants exhibited a 30% decrease (P < 0.05) in cell growth and an enhanced differentiation phenotype. Increased PKC-delta expression induced a significant G0/G1 arrest, inhibited anchorage-independent growth (50%, P < 0.05), and caused a 2-fold increase in apoptosis (P < 0.05). CONCLUSIONS: Our studies show that increased expression of PKC-delta inhibits anchorage-dependent and -independent growth, while inducing cellular differentiation and limiting survival of this human colon cancer cell line.

The role of vitamin D in normal and pathologic processes in the colon.

Vitamin D(3) metabolites and analogues have recently been shown to play an important role in the regulation of a number of important cellular processes, including proliferation, differentiation, and apoptosis, in addition to their established roles in mineral homeostasis. The actions of these secosteroids involve both rapid, nongenomic effects and genomic effects; the latter mediated via the vitamin D receptor and other transcription factors. Their effects have been described in a variety of cell types, including normal and malignant colonocytes. This article summarizes the rapid and genomic actions of vitamin D(3) metabolites and analogues on normal and pathologic processes in the colon, with particular emphasis on the potential of these secosteroids to prevent colon cancer.

A vitamin D3 analog induces a G1-phase arrest in CaCo-2 cells by inhibiting cdk2 and cdk6: roles of cyclin E, p21Waf1, and p27Kip1.

Previous studies by our laboratory have shown that a noncalcemic fluorinated analog of 1alpha,25-dihydroxyvitamin D3, 1alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholcal ciferol (F6-D3), significantly reduced the frequency of colonic adenomas and completely abolished the development of colonic adenocarcinomas in rats treated with azoxymethane. The mechanisms involved in this analog's chemopreventive actions, however, remain unclear. In the present study, we now show that although both 1alpha,25-dihydroxyvitamin D3 and F6-D3 inhibited the proliferation of CaCo-2 cells, a human colonic adenocarcinoma cell line, by increasing their doubling times, only F6-D3 caused an arrest of these cells in the G1 phase of their cell cycle. This arrest was accompanied by an increase in the expression of the cyclin-dependent kinase (cdk) inhibitor proteins, p2Waf1 and p27Kip1, which served to decrease the activity of cyclin-dependent kinase 2 and cyclin-dependent kinase 6, whereas the expression and phosphorylation of pRB were unchanged. In contrast to the increased expression of these cdk inhibitors, the expression of cyclin E was decreased, which further inhibited the activity of cyclin-dependent kinase 2. Collectively, the inhibition of these cyclin-dependent kinases served to arrest the CaCo-2 cells, independent of changes in pRB. Furthermore, antibody neutralization studies suggest that transforming growth factor-beta may mediate the coassociations between cdk2 and p27Kip1 and cyclin E induced by F6-D3. These data indicate that cell cycle arrest may, at least in part, underlie the chemopreventive actions of F6-D3 observed in the azoxymethane model of colon cancer. Furthermore, if the antiproliferative action observed in CaCo-2 cells also occurs in human colonic epithelium, F6-D3 may have chemopreventive potential against human colon cancer, as well.

Mutational and nonmutational activation of p21ras in rat colonic azoxymethane-induced tumors: effects on mitogen-activated protein kinase, cyclooxygenase-2, and cyclin D1.

Azoxymethane (AOM)-induced colonic carcinogenesis involves a number of mutations, including those in the K-ras gene and CTNNB1, that codes for beta-catenin. Prior in vitro studies have also demonstrated that wild type p21(K-ras) can be activated by epigenetic events. We identified 15 K-ras mutations in 14 of 84 AOM-induced colonic tumors by three independent methods. By single strand conformational polymorphism, we also observed mutations in 22 of 68 tumors in exon 3 of CTNNB1. A highly sensitive method was then used to measure p21ras activation levels. All tumors assayed possessing K-ras mutations had significantly higher p21ras activation levels (8.8 +/- 1.5%; n = 13) compared with that of control colon (3.7 +/- 0.4; n = 6; P < 0.05) or tumors without such mutations (4.2 +/- 0.4%; n = 70; P < 0.05). Among tumors with wild-type K-ras, there was a subset of tumors (18 of 70) that had significantly higher p21ras activation levels (8.0 +/- 0.9%; n = 18) compared with control colons. In three of four tumors examined with activated wild-type p21ras, we observed increased c-erbB-2 receptor expression and decreased Ras-GAP expression. In contrast, only one of eight tumors examined with wild-type ras and nonactivated p21ras demonstrated these alterations. Mitogen-activated protein kinase (MAPK) activation and cyclooxygenase-2 (COX-2) expression were increased in tumors with mutated or activated wild-type p21ras, compared with their nonactivated counterparts. Although beta-catenin mutations did not alter COX-2 expression or MAPK activity, mutations in either K-ras or beta-catenin significantly increased cyclin D1 expression. In contrast, in tumors with wild-type but activated p21-ras, cyclin D1 expression was not enhanced. Thus, the spectrum of changes in MAPK, COX-2, and cyclin D1 is distinct among tumors with ras or beta-catenin mutations or nonmutational activation of p21ras.

Inhibition of O(6)-methylguanine-DNA methyltransferase increases azoxymethane-induced colonic tumors in rats.

Azoxymethane (AOM) causes O(6)-methylguanine adduct formation which leads to G-->A transitions. Their repair is carried out by O(6)-methylguanine-DNA methyltransferase (MGMT). To evaluate the importance of this repair event in AOM-induced carcinogenesis, we examined the effect of O(6)-benzylguanine (BG), a potent inhibitor of MGMT, on colonic tumor development. Rats were treated weekly for 2 weeks at 0 and 24 h with BG (60 mg/kg body wt i.p.) or vehicle (40% polyethylene glycol, PEG-400), followed 2 h after the first dose of BG with AOM (15 mg/kg body wt) or vehicle (saline) i.p. Rats were killed 35 weeks later and tumors harvested and DNA extracted. In the AOM-treated groups, BG caused a significant increase in tumor incidence with tumors in 65.9%, versus 30.8% in the AOM/PEG-treated group (P < 0.05). In the BG/AOM group there was also a significant increase in tumor multiplicity, with 2.3 tumors/tumor-bearing rat, versus 1.6 tumors/tumor- bearing rat in the AOM/PEG group (P < 0.05). Since O(6)-methylguanine adducts can cause activating mutations in the K-ras and beta-catenin genes, we examined the effects of BG on these mutations. In the BG group there were seven mutations in codon 12 or 13 of exon 1 of the K-ras gene in 51 tumors examined, compared with no K-ras mutations in 17 tumors analyzed in the AOM/PEG group (P = 0.12). In the BG/AOM group there were 10 mutations in exon 3 of the beta-catenin gene among 48 tumors evaluated, compared with six mutations in 16 tumors analyzed in the PEG/AOM group (P = 0.16). In summary, MGMT inhibition increases AOM-induced colonic tumor incidence and multiplicity in rats.

1,25-Dihydroxyvitamin D(3) stimulates activator protein-1-dependent Caco-2 cell differentiation.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is a potential chemopreventive agent for human colon cancer. We have reported that 1,25(OH)(2)D(3) specifically activated protein kinase C-alpha (PKC-alpha) 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)(2)D(3) activated AP-1 in CaCo-2 cells and, if so, by what mechanisms? 1,25(OH)(2)D(3) 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)(2)D(3) 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)(2)D(3) 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)(2)D(3) was mediated by ERK and JNK. Using a specific inhibitor of the Ca(2+)-dependent PKC isoforms, Gö6976, and CaCo-2 cells stably transfected with antisense PKC-alpha cDNA, demonstrated that PKC-alpha mediated the AP-1 activation induced by this secosteroid. Inhibition of JNK activation or c-Jun protein expression significantly reduced 1, 25(OH)(2)D(3)-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)(2)D(3) stimulated AP-1 activation in CaCo-2 cells by a PKC-alpha- and JNK-dependent mechanism leading to increases in cellular differentiation.

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 and TPA activate phospholipase D in Caco-2 cells: role of PKC-alpha.

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)2D3 activated PKC-alpha, but not PKC-beta1, -betaII, -delta, or -zeta, whereas TPA activated PKC-alpha, -beta1, and -delta. Chronic treatment with TPA (1 microM, 24 h) significantly reduced the expression of PKC-alpha, -betaI, and -delta and markedly reduced the ability of 1,25(OH)2D3 or 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)2D3 or TPA. Treatment with 12-deoxyphorbol-13-phenylacetate-20-acetate, which specifically activates PKC-betaI and -betaII, however, failed to stimulate PLD. In addition, the activation of PLD by 1,25(OH)2D3 or TPA was markedly reduced or accentuated in stably transfected cells with inhibited or amplified PKC-alpha expression, respectively. Taken together, these observations indicate that PKC-alpha is intimately involved in the stimulation of PLD in Caco-2 cells by 1,25(OH)2D3 or TPA.

1,25-dihydroxyvitamin D3 but not TPA activates PLD in Caco-2 cells via pp60(c-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-alpha (PKC-alpha) 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 pp60(c-src) have been implicated in PLD activation. We therefore determined whether these signal transduction elements were involved in PLD stimulation by 1,25(OH)2D3 or 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 pp60(c-src) and treatment with PP1, a specific inhibitor of the pp60(c-src) family, blocked the ability of 1,25(OH)2D3 to activate PLD. Pretreatment of cells with pertussis toxin (PTx) markedly reduced the stimulation of PLD by either agonist. PTx, moreover, inhibited the stimulation of pp60(c-src) and PKC-alpha by 1,25(OH)2D3. PTx did not, however, block the membrane translocation of RhoA induced by 1,25(OH)2D3 or inhibit the stimulation of PKC-alpha by TPA. These findings, taken together with those of the accompanying paper, indicate that although 1,25(OH)2D3 and TPA each activate PLD in Caco-2 cells in part via PKC-alpha, their stimulation of PLD differs in a number of important aspects, including the requirement for pp60(c-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)2D3 and TPA to induce the stimulation of PLD may potentially underlie differences in the physiological effects of these agents in Caco-2 cells.

Expression of G protein alpha subunits in normal rat colon and in azoxymethane-induced colonic neoplasms.

BACKGROUND & AIMS: Heterotrimeric G proteins are important in growth-regulating signal transduction. The aim of this study was to characterize the relative expression of G protein alpha subunits in rat colonocytes, colonocyte antipodal plasma membranes, and colonic neoplasms. METHODS: Antipodal plasma membranes were prepared from isolated colonocytes. Azoxymethane was administered to rats to induce colonic neoplasms. K-ras mutations in the neoplasms were determined by oligonucleotide hybridization and confirmed by primer mediated-restriction fragment length polymorphism. Colonocyte and tumor homogenates or membranes were probed for Galpha subunits by Western blotting with isoform-specific antibodies. RESULTS: The expressions of Galphai2, alphai3, and alphaq/11 were significantly enriched in the basolateral compared with brush border fraction of colonic antipodal plasma membranes. In neoplasms without K-ras mutations, the expression of Galphai2 increased 4-fold, Galphas(long) increased 2.5-fold, and Galphai3 increased 1.5-2-fold. Expression did not differ among tumor grades. K-ras mutations were associated with lowered expression of G proteins, especially Galphao. CONCLUSIONS: In colonocytes, Galpha subunits are localized primarily in basolateral plasma membranes. The increased expressions of Galphai2 and, to a lesser degree, Galphai3 and Galphas(long) in tumors was independent of tumor grade but was modulated by the presence of K-ras mutations.

1,25-Dihydroxyvitamin D3 targets PKC-betaII but not PKC-alpha to the basolateral plasma membranes of rat colonocytes.

Prior studies by our laboratory have shown that 1, 25-dihydroxyvitamin D3 activated PKC-alpha, but not PKC-delta, -epsilon, or -zeta, in normal rat colonocytes. In the present studies we demonstrate for the first time that this secosteroid also activated PKC-betaII, another DAG- and Ca2+-dependent PKC isoform recently shown to be present in these cells. Moreover, this activation of PKC-betaII by 1,25-dihydroxyvitamin D3 treatment of isolated colonocytes was shown to be lost in cells from vitamin D-deficient rats and, at least partially, restored by repleting these animals with this secosteroid for 7 days. Under basal conditions, the expression of PKC-alpha and -betaII in brush-border membranes was comparable to their respective expression in basolateral plasma membranes of rat colonocytes. In contrast, the expression of PKC-delta was significantly greater in brush-border membranes, whereas PKC-epsilon and -zeta were enriched in the basolateral plasma membranes. Furthermore, 1,25-dihydroxyvitamin D3 specifically induced the translocation of PKC-betaII, but not PKC-alpha, to the basolateral, but not brush-border plasma membranes of rat colonocytes, via a pp60(c-src)-dependent mechanism.

Enrichment of the more hydrophilic bile acid ursodeoxycholic acid in the fecal water-soluble fraction after feeding to rats with colon polyps.

We recently showed that feeding the cytoprotective bile acid ursodeoxycholic acid (UDCA) to rats resulted in significant reduction in polyps and especially cancers, both in number and size (D. L. Earnest et al., Cancer Res., 54: 5071-5074, 1994). Because fecal secondary bile acids [particularly deoxycholic acid (DCA)] are considered to promote formation of colon adenomas and cancer, we have now attempted to find a relationship between polyp reduction and fecal secondary bile acids after feeding UDCA to these rats. We examined the fecal bile acids in rats with polyps and compared them with fecal bile acids in control rats and also determined the bile acid composition in fecal aqueous phase, which is in direct contact with the colon epithelium and may be physiologically more active. Treatment with azoxymethane did not significantly alter fecal bile acid composition in the rats. Cholic acid feeding resulted in greatly increased proportions of DCA (82% of total bile acids versus 18% in control rats). On the other hand, UDCA feeding significantly reduced the proportion of fecal DCA (2% in control rats fed UDCA and 3% in rats also treated with azoxymethane). In control rats, 96% of the bile acids were present in the water-insoluble fraction and 4% in the water-soluble fraction. The major insoluble bile acids included DCA and hyodeoxycholic acid (73% of total bile acids). In contrast, the muricholic acids were concentrated in the soluble fraction (37%). When 0.4% UDCA was added to the diet, lithocholic acid increased in the insoluble fraction (40 versus 1%), but the hydrophilic UDCA and muricholic acids were enriched in the water-soluble fraction (37 and 43%, respectively). Thus, the hydrophobic bile acids were distributed predominantly in the water-insoluble fraction, whereas the hydrophilic bile acids were distributed preferentially in the water-soluble fraction. These data suggest that UDCA may prevent colon tumors and polyps by countering the toxic effect of DCA and enhancing the possible cytoprotective effects of UDCA and muricholic acids in the water-soluble fraction in the feces of rat.

Protein kinase C alpha modulates growth and differentiation in Caco-2 cells.

BACKGROUND & AIMS: Caco-2 cells have been used extensively to elucidate events involved in intestinal cell proliferation and differentiation. Because individual isoforms of protein kinase C (PKC) and p21waf1, a cyclin-dependent kinase inhibitor, may regulate these processes, their role(s) on the growth and differentiation of Caco-2 cells were assessed. METHODS: Protein abundance and subcellular distribution of several PKC isoforms, as well as the expression of p21waf1, were examined in preconfluent and postconfluent cells. RESULTS: In cells at confluence (approximately 7 days postplating) and during their postconfluent phase (up to 20 days postplating), both total protein expression of PKC-alpha and its particulate distribution increased compared with their 3-day postplated counterparts. These findings were in agreement with those obtained by immunocytochemistry of PKC-alpha. In contrast, neither the total expression nor the subcellular distribution of PKC-betaI, -betaII, -delta, or -zeta changed significantly during these time periods. In addition, the expression of p21waf1, which can be induced by PKC-alpha, increased in postconfluent cells. CONCLUSIONS: PKC-alpha, but not other isoforms of PKC, may modulate the proliferation and differentiation of Caco-2 cells. This regulation appears to be mediated, at least in part, via a mechanism involving p21waf1.

Decreased PKC-alpha expression increases cellular proliferation, decreases differentiation, and enhances the transformed phenotype of CaCo-2 cells.

Previous studies have shown that PKC-alpha protein expression is decreased in sporadic human colon cancers, as well as in colonic tumors of rats induced by chemical carcinogens. To elucidate the potential role of PKC-alpha on several phenotypic characteristics of colon cancer cells, we have transfected cDNAs for PKC-alpha in sense or antisense orientations into CaCo-2 cells, a human colonic adenocarcinoma cell line. Transfected clones were isolated that demonstrated approximately 3-fold increases (sense transfectants) and approximately 95% decreases (antisense transfectants) in PKC-alpha expression with no significant alterations in other PKC isoforms. Transfection of CaCo-2 cells with PKC-alpha in the antisense orientation resulted in enhanced proliferation and decreased differentiation, as well as in a more aggressive transformed phenotype compared with empty vector-transfected control cells. In contrast, cells transfected with PKC-alpha cDNA in the sense orientation demonstrated decreased proliferation, enhanced differentiation, and an attenuated tumor phenotype compared with these control cells. These data show that alterations in the expression of PKC-alpha induce changes in the proliferation, differentiation, and tumorigenicity of CaCo-2 cells. Furthermore, these findings indicate that loss of PKC-alpha expression in sporadic human and chemically induced colonic cancers may confer a relative growth advantage during colonic malignant transformation.

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.

Inverse relationship between membrane lipid fluidity and activity of Na+/H+ exchangers, NHE1 and NHE3, in transfected fibroblasts.

This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants of an NHE-deficient fibroblast, we analyzed each isoform separately. An increase in membrane fluidity resulted in a decrease of approximately 50% in the specific activities of both NHE1 and NHE3. Only Vmax was affected; KNa was unchanged. This effect was specific, as Na+, K+, ATPase activity was slightly stimulated. Inhibition of NHE1 and NHE3 was reversible and de novo protein synthesis was not required to restore NHE activity after washout of fluidizer. Inhibition kinetics of NHE1 by amiloride, 5-(N,N-dimethyl)amiloride (DMA), 5-(N-hexamethyl)amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were largely unchanged. Half-maximal inhibition of NHE3 was also reached at approximately the same concentrations of amiloride and analogues in control and benzyl alcohol treated, suggesting that the amiloride binding site was unaffected. Inhibition of vesicular transport by incubation at 4 degrees C augmented the benzyl alcohol inhibition of NHE activity, suggesting that the fluidizer effect does not solely involve vesicle trafficking. In summary, our data demonstrate that the physical state of membrane lipids (fluidity) influences Na+/H+ exchange and may represent a physiological regulatory mechanism of NHE1 and NHE3 activity.

Transport of n-butyrate into human colonic luminal membrane vesicles.

Human colonic short-chain fatty acid (SCFA) absorption is associated with increased luminal pH and HCO3- and enhanced Na+ absorption. Therefore, the mechanism of colonic SCFA transport, its dependence on Na+ and HCO3- and interactions with Cl-/HCO3- and Na+/H+ exchangers were characterized. Luminal membrane vesicles (LMV) isolated by divalent cation precipitation from organ donor colons were used for n-butyrate transport. Uptake of n-butyrate into the human colonic LMV was minimal even in the presence of an inward pH gradient, but an outward HCO3- gradient significantly increased uptake rates. HCO3(-)-stimulated butyrate uptake was saturable with an apparent Michaelis constant of 1.5 +/- 0.2 mM and maximal velocity of 105 +/- 3 nmol.mg protein-1.3 s-1. Intravesicular butyrate resulted in trans-stimulation of n-[1-14C]butyrate uptake. Butyrate uptake was inhibited approximately 25-40% by C2-C5 SCFAs and approximately 40% by niflumic acid. Butyrate uptake was not affected by extravesicular Na+, and 22Na uptake was unaltered by extravesicular butyrate. Butyrate uptake was independent of extra- or intravesicular CI-, and butyrate loading produced no changes in 36Cl uptake. We conclude that the predominant mechanism of n-butyrate transport across the human colonic luminal membrane appears to be via a HCO3-/SCFA antiport system independent of Cl-/HCO3- exchange and Na+ transport.