We reported previously that protein kinase C␣ (PKC␣), a negative regulator of cell growth in the intestinal epithelium, inhibits cyclin D1 translation by inducing hypophosphorylation/activation of the translational repressor 4E-BP1. The current study explores the molecular mechanisms underlying PKC/PKC␣-induced activation of 4E-BP1 in IEC-18 nontransformed rat ileal crypt cells. PKC signaling is shown to promote dephosphorylation of Thr 45 and Ser 64 on 4E-BP1, residues directly involved in its association with eIF4E. Consistent with the known role of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway in regulation of 4E-BP1, PKC signaling transiently inhibited PI3K activity and Akt phosphorylation in IEC-18 cells. However, PKC/PKC␣-induced activation of 4E-BP1 was not prevented by constitutively active mutants of PI3K or Akt, indicating that blockade of PI3K/Akt signaling is not the primary effector of 4E-BP1 activation. This idea is supported by the fact that PKC activation did not alter S6 kinase activity in these cells. Further analysis indicated that PKC-mediated 4E-BP1 hypophosphorylation is dependent on the activity of protein phosphatase 2A (PP2A). PKC signaling induced an ϳ2-fold increase in PP2A activity, and phosphatase inhibition blocked the effects of PKC agonists on 4E-BP1 phosphorylation and cyclin D1 expression. H 2 O 2 and ceramide, two naturally occurring PKC␣ agonists that promote growth arrest in intestinal cells, activate 4E-BP1 in PKC/PKC␣-dependent manner, supporting the physiological significance of the findings. Together, our studies indicate that activation of PP2A is an important mechanism underlying PKC/ PKC␣-induced inhibition of cap-dependent translation and growth suppression in intestinal epithelial cells. Members of the protein kinase C (PKC)3 family of signal transduction molecules have been implicated in regulation of fundamental cellular processes, including cell growth and cell cycle progression, differentiation, survival/apoptosis, protein translation, membrane trafficking, receptor desensitization, and migration (1-10). Increasing evidence supports a key role for PKC signaling in the maintenance of intestinal homeostasis (1,11,12), and aberrations in PKC expression and function occur early during intestinal tumorigenesis (13). Previous studies from this laboratory have demonstrated that PKC/PKC␣ signaling triggers a program of cell cycle withdrawal in intestinal epithelial cells characterized by rapid down-regulation of cyclin D1, increased expression of Cip/Kip cyclin-dependent kinase inhibitors, and activation of the growth suppressor function of members of the pocket protein family (14, 15). Downregulation of cyclin D1 precedes various hallmark events of cell cycle exit, pointing to the importance of regulation of this cyclin in PKC-mediated growth arrest in intestinal epithelial cells.Analysis of the mechanisms underlying PKC/PKC␣-mediated down-regulation of cyclin D1 identified PKC␣ as a negative regulator of cyclin D1 translation (7). PKC/PKC␣ signaling activates t...

Alterations in PKC isozyme expression and aberrant induction of cyclin D1 are early events in intestinal tumorigenesis. Previous studies have identified cyclin D1 as a major target in the antiproliferative effects of PKCα in non-transformed intestinal cells; however, a link between PKC signaling and cyclin D1 in colon cancer remained to be established. The current study further characterized PKC isozyme expression in intestinal neoplasms and explored the consequences of restoring PKCα or PKCδ in a panel of colon carcinoma cell lines. Consistent with patterns of PKC expression in primary tumors, PKCα and δ levels were generally reduced in colon carcinoma cell lines, PKCβII was elevated and PKCε showed variable expression, thus establishing the suitability of these models for analysis of PKC signaling. While colon cancer cells were insensitive to the effects of PKC agonists on cyclin D1 levels, restoration of PKCα downregulated cyclin D1 by two independent mechanisms. PKCα expression consistently (a) reduced steady-state levels of cyclin D1 by a novel transcriptional mechanism not previously seen in non-transformed cells, and (b) re-established the ability of PKC agonists to activate the translational repressor 4E-BP1 and inhibit cyclin D1 translation. In contrast, PKCδ had modest and variable effects on cyclin D1 steady state levels and failed to restore responsiveness to PKC agonists. Notably, PKCα expression blocked anchorage-independent growth in colon cancer cells via a mechanism partially dependent on cyclin D1 deficiency, while PKCδ had only minor effects. Loss of PKCα and effects of its re-expression were independent of the status of the APC/β-catenin signaling pathway or known genetic alterations, indicating that they are a general characteristic of colon tumors. Thus, PKCα is a potent negative regulator of cyclin D1 expression and anchorage-independent cell growth in colon tumor cells, findings that offer important perspectives on the frequent loss of this isozyme during intestinal carcinogenesis.

Increasing evidence supports a role for PKC␣ in growth arrest and tumor suppression in the intestinal epithelium. In contrast, the Id1 transcriptional repressor has pro-proliferative and tumorigenic properties in this tissue. Here, we identify Id1 as a novel target of PKC␣ signaling. Using a highly specific antibody and a combined morphological/biochemical approach, we establish that Id1 is a nuclear protein restricted to proliferating intestinal crypt cells. A relationship between PKC␣ and Id1 was supported by the demonstration that (a) down-regulation of Id1 at the crypt/villus junction coincides with PKC␣ activation, and (b) loss of PKC␣ in intestinal tumors is associated with increased levels of nuclear Id1. Manipulation of PKC␣ activity in IEC-18 nontransformed intestinal crypt cells determined that PKC␣ suppresses Id1 mRNA and protein via an Erk-dependent mechanism. PKC␣, but not PKC␦, also inhibited Id1 expression in colon cancer cells. Id1 was found to regulate cyclin D1 levels in IEC-18 and colon cancer cells, pointing to a role for Id1 suppression in the antiproliferative/tumor suppressive activities of PKC␣. Notably, Id1 expression was elevated in the intestinal epithelium of PKC␣-knock-out mice, confirming that PKC␣ regulates Id1 in vivo. A wider role for PKC␣ in control of inhibitor of DNA binding factors is supported by its ability to downregulate Id2 and Id3 in IEC-18 cells, although their suppression is more modest than that of Id1. This study provides the first demonstrated link between a specific PKC isozyme and inhibitor of DNA binding factors, and it points to a role for a PKC␣ 3 Erk ٜ Id1 3 cyclin D1 signaling axis in the maintenance of intestinal homeostasis.The intestinal epithelium is a continually self-renewing tissue, organized into well defined proliferative and functional compartments (1). Maintenance of tissue integrity relies on tight control of the balance between proliferative activity, differentiation, and apoptosis. Both positive and negative growth regulatory signaling pathways have been implicated in orchestrating the renewal process in this tissue, and disruption of these pathways and/or their downstream targets results in various diseases, including cancer. Increasing evidence points to the PKC enzyme system as a key regulator of intestinal homeostasis (2-6). PKC is a family of phospholipid-dependent serine-threonine kinases, consisting of at least 10 isozymes that act as central players in signal transduction. Members of the family can have tumor promoting (e.g. PKC␤II, -⑀, and -) or tumor suppressive (e.g. PKC␣ and -␦) activity, dependent on the isozyme and tissue context (2,5,7,8). Although several PKC isozymes have been implicated in the regulation of intestinal homeostasis (e.g. PKC␣, -␤II, -␦, -⑀, and -), increasing evidence points to PKC␣ as a key negative regulator of proliferation and tumorigenesis in this tissue (4, 9, 10).PKC␣ is activated precisely at the point of growth arrest in the crypts of both the small intestine and colon (11, 12). Consistent with a role i...

Int22h1/Int22h2‐mediated Xq28 duplication syndrome is a relatively new X‐linked intellectual disability syndrome, arising from duplications of the subregion flanked by intron 22 homologous regions 1 and 2 on the q arm of chromosome X. Its primary manifestations include variable cognitive deficits, distinct facial dysmorphia, and neurobehavioral abnormalities that mainly include hyperactivity, irritability, and autistic behavior. Affected males are hemizygous for the duplication, which explains their often more severe manifestations compared with heterozygous females. In this report, we describe the cases of nine individuals recently identified having the syndrome, highlighting unique and previously unreported findings of this syndrome. Specifically, we report for the first time in this syndrome, two cases with de novo duplications, three receiving prenatal diagnosis with the syndrome, and three others having atypical versions of the duplication. Among the latter, one proband has a shortened version spanning only the centromeric half of the typical duplication, while the other two cases have a nearly identical length duplication as the classical duplication, with the exception that their duplication's breakpoints are telomerically shifted by about 0.2 Mb. Finally, we shed light on two new manifestations in this syndrome, vertebral anomalies and multiple malignancies, which possibly expand the phenotypic spectrum of the syndrome.

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