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.

Expression of a recombinant apolipoprotein(a) in HepG2 cells. Evidence for intracellular assembly of lipoprotein(a).

Apolipoprotein(a) (apo(a)), a large glycoprotein with extensive homology to plasminogen, forms a complex with apolipoprotein B100 (apoB100), which circulates in human plasma in the form of lipoprotein(a) (Lp(a)). Evidence indicates that the association of apo(a) with apoB100 occurs in the extracellular environment. We have reevaluated the possibility that apo(a)-B100 association can also occur as an intracellular event through studies with HepG2 cells stably transfected with an apo(a) minigene. Several lines of evidence support this possibility. First, continued Lp(a) production was demonstrated following incubation of transfected HepG2 cells with anti-apo(a) antisera, conditions that effectively block the fluid-phase association of apo(a) and apoB100 in vitro. Second, an apo(a)-B100 complex was detectable in Western blot analyses of transfected HepG2 lysates following immunoprecipitation with anti-apo(a) antisera. These studies incorporated precautions to eliminate cell-surface attachment of preformed apo(a)-B100 complexes to the low density lipoprotein receptor and were conducted in the presence of the lysine analog epsilon-aminocaproic acid, which precludes apo(a)-B100 association occurring during the isolation and analyses. Third, the presence of an intracellular apo(a)-B100 complex was demonstrated in lipoproteins isolated from microsomal contents. Of particular significance was the observation that this complex contained the precursor form of apo(a), which is not secreted, in addition to the mature, recombinant form. Finally, direct evidence was provided for the synthesis of a precursor form of apo(a) in a nascent intracellular complex with apoB100 following treatment of transfected HepG2 cells with brefeldin A plus N-acetyl-leucyl-leucyl-norleucinal. Taken together, these data suggest that apo(a)-B100 association can occur as an intracellular event in a human hepatoma-derived cell line, raising important implications for the regulation of Lp(a) secretion from human liver.

Developmental regulation of the catalytic subunit of the apolipoprotein B mRNA editing enzyme (APOBEC-1) in human small intestine.

Apolipoprotein (apo) B mRNA editing is a site-specific cytidine deamination reaction responsible for the production of apoB-48 in mammalian small intestine. This process is mediated by an enzyme complex that includes the catalytic subunit, APOBEC-1. In the present study, it is shown that the developmental regulation of apoB mRNA editing in fetal human small intestine is closely mirrored by accumulation of APOBEC-1 mRNA. Similar results were obtained using Caco-2 cells, the data further suggesting that culture of these cells under conditions previously shown to promote differentiation produce an earlier and more marked induction of APOBEC-1 mRNA abundance. Complementary analysis of APOBEC-1 protein accumulation using immunocytochemical localization reveals its appearance to be temporally coordinated with the accumulation of APOBEC-1 mRNA and its distribution to be confined to villus-associated enterocytes. Previous studies demonstrated a close temporal association between the development of triglyceride synthesis and apoB mRNA editing in the rat liver and small intestine. Analysis of fatty acid CoA ligase, monoacylglycerol acyltransferase, and diacylglycerol acyltransferase activity in preparations of human liver and small intestine demonstrates activity of all three enzymes in the late first and early second trimester, suggesting that certain aspects of complex lipid biosynthesis in the human fetal small intestine and liver are regulated developmentally. The cues that modulate the post-transcriptional regulation of fetal human small intestinal apoB gene expression may thus include both temporal programming and events related to the emergence of lipid transport capability.

Tissue-specific, developmental and nutritional regulation of the gene encoding the catalytic subunit of the rat apolipoprotein B mRNA editing enzyme: functional role in the modulation of apoB mRNA editing.

Apolipoprotein B (apoB) mRNA editing, a posttranscriptional site-specific cytidine deamination reaction, is mediated by a protein complex of which the catalytic component (REPR) has recently been cloned. REPR mRNA was demonstrated by RNase protection at highest abundance in small intestine and colon but the transcript was detectable in all tissues examined including kidney, spleen, lung, liver, and ovary. ApoB mRNA was found predominantly in the liver and small intestine but low levels were detected in all adult tissues examined and found to be variably (29-86% TAA) edited. In addition, S100 extracts prepared from spleen and kidney were competent to edit an apoB RNA template in vitro, suggesting that the entire apoB mRNA editing complex is present and functionally active in these tissues. In situ hybridization demonstrated REPR mRNA to be distributed along the entire villus-crypt axis, while apoB mRNA distribution did not extend into the crypts. In the liver, both apoB and REPR mRNA were detected in all cells of the hepatic lobule without an apparent gradient of expression. REPR mRNA was found in the red pulp of the spleen and in the superficial crypt cells of the colon. This distribution of REPR mRNA was recapitulated by immunocytochemical localization of the protein within these tissues. Finally, the developmental and nutritional modulation of REPR was examined in relation to endogenous apoB mRNA editing. Small intestinal apoB mRNA editing was found to undergo a developmentally regulated increase beginning at gestational day 20, preceding a developmental increase in REPR mRNA abundance. Additionally, hepatic and kidney apoB mRNA editing both revealed a temporal dissociation from alterations in REPR mRNA abundance. By contrast, adult rats subjected to fasting and refeeding a high carbohydrate diet, demonstrated concordant modulation of endogenous apoB mRNA editing and REPR mRNA abundance (r = 0.92, P < 0.001). Taken together, the data demonstrate that REPR and other components of the rat apoB mRNA editing complex are widely distributed and undergo distinct developmental and metabolic regulation that interact to regulate apoB mRNA editing in a tissue-specific manner.

Molecular cloning of a human small intestinal apolipoprotein B mRNA editing protein.

Mammalian small intestinal apolipoprotein B (apo B) mRNA undergoes posttranscriptional cytidine deamination with the production of an in frame stop codon and the translation of apo B48. We have isolated a cDNA from human jejunum which mediates in vitro editing of a synthetic apo B RNA template upon complementation with chicken intestinal S100 extracts. The cDNA specifies a 236 residue protein which is 69% identical to the apo B mRNA editing protein (REPR) cloned from rat small intestine [Teng, B., Burant, C. F. and Davidson, N. O. (1993) Science 260, 1816-1819] and which, by analogy, is referred to as HEPR. HEPR does not contain the carboxyl-terminus leucine zipper motif identified in REPR but contains consensus phosphorylation sites as well as the conserved histidine and both cysteine residues identified as a Zn2+ binding motif in other cytidine deaminases. The distribution of HEPR mRNA was predominantly confined to the adult small intestine with lower levels detectable by reverse-transcription polymerase chain reaction amplification in the stomach, colon and testis. These differences in the structure and distribution of the human as compared to the rat apo B mRNA editing protein suggest an important evolutionary adaptation in the mechanisms restricting apo B48 production to the small intestine.

Effects of 1,25-dihydroxyvitamin D3 on proliferation and differentiation of Caco-2 cells.

The effect of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or calcitriol, on the proliferation and differentiation of Caco-2 cells was studied. Vitamin D receptor mRNA was detected in both pre- and postconfluent cells, and its abundance was unchanged with time and in response to calcitriol. 1,25-(OH)2D3-binding activity increased during differentiation, but there was no difference in binding between 1,25-(OH)2D3-treated and control cells. 1,25-(OH)2D3 caused a dose-dependent reduction in proliferation, as assessed by [3H]thymidine incorporation and DNA content. 1,25-(OH)2D3 significantly enhanced the normal rise in alkaline phosphatase activity during differentiation and increased alkaline phosphatase mRNA abundance. In contrast, 1,25-(OH)2D3 inhibited the normal rise in sucrase-isomaltase activity and the corresponding mRNA level, although the inhibition occurred after the initial period of cell differentiation (> 10 days postplating). Morphological analysis demonstrated that by day 12 postplating, 1,25-(OH)2D3 increased the mean dome diameter and microvillus length and density. Although 1,25-(OH)2D3 decreases the proliferation of Caco-2 cells and enhances certain parameters of differentiation, not all brush-border hydrolases respond in a similar fashion, making it necessary to interpret with caution their individual use as markers of differentiation.

Tissue and cell-specific patterns of expression of rat liver and intestinal fatty acid binding protein during development and in experimental colonic and small intestinal adenocarcinomas.

BACKGROUND: Mammalian small intestinal and colonic epithelium express members of a multigene family of hydrophobic ligand binding proteins of which liver and intestinal fatty acid binding protein represent among the most abundant intestinal gene products. These proteins are expressed in a cell- and region-specific manner and emerge in a temporally distinctive pattern. EXPERIMENTAL DESIGN: We have studied expression of these genes in the small intestine and colon of rats treated with 1,2-dimethylhydrazine since these animals develop a predictable pattern of both small and large bowel cancers. Studies were also undertaken in fetal and neonatal small intestine and colon. RESULTS: There was a 10- and 50-fold decrease, respectively, in mRNA abundance for intestinal and liver fatty acid binding protein in RNA from colon cancers compared with either uninvolved or control RNA. Immunocytochemical analysis revealed decreased staining for both proteins within their normal distribution together with ectopic clusters of cells reactive for liver fatty acid binding protein within colonic tumors. A more striking mosaic of immunocytochemical staining for both liver and intestinal fatty acid binding protein was found in small intestinal adenocarcinomas. Similar mosaic patterns of immunocytochemical staining were transiently detectable in rat fetal small intestine and neonatal colon. CONCLUSIONS: The region- and cell-specific expression of these genes, which may be linked temporally to events in intestinal differentiation, are subject to disruption in a cell-specific manner in the transformed, or dedifferentiated, phenotype.