Negative Regulator Of Cellular Proliferation Research Articles

Cyclase-associated protein 1 is a key negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells.

Adenylyl cyclase-associated protein (CAP) is a highly conserved protein. Previous reports have suggested that CAP1 may be a negative regulator of cellular proliferation, migration, and adhesion and the development of cell carcinomas. The molecular mechanism of CAP1 regulation of downstream pathways, as well as how CAP1 is regulated by environmental stimuli and upstream signalling, is not well understood. In this present study, we assessed the role of CAP1 in milk synthesis and proliferation of bovine mammary epithelial cells. Using gene overexpression and silencing methods, CAP1 was found to negatively regulate milk synthesis and proliferation of cells via the PI3K-mTOR/SREBP-1c/Cyclin D1 signalling pathway. Hormones, such as prolactin and oestrogen, and amino acids, such as methionine and leucine, stimulate MMP9 expression and trigger CAP1 degradation, and thus, abrogate its inhibition of synthesis of milk protein, fat, and lactose by and proliferation of bovine mammary epithelial cells. The results of our study help deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in characterizing the molecular mechanisms of CAP1. Previous reports have suggested that CAP1 is a negative regulator of cellular proliferation and anabolism, but the molecular mechanisms are largely unknown. In this present study, we identified CAP1 as a negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells. Our results will deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in exploring the molecular mechanisms of CAP1.

Prohibitin: targeting peptide coupled to ovarian cancer, luteinization and TGF-\u03b2 pathways

BackgroundOvarian epithelial tumor (OET) is a silent disease of late diagnosis and poor prognosis. Currently treatment options are limited and patient response to treatment is difficult to predict so there is a serious need to delineate the real pathogenesis to predict tumour prognosis. Prohibitin (PHB) is an evolutionarily protein that regulates the cell cycle. TGF-β has been shown to be a positive and negative regulator of cellular proliferation and differentiation.The present study provides an overview on the role played by PHB1, TGF-β and LH in ovarian cancer.MethodsThe study was conducted on 60 patients with ovarian tumors (benign, borderline and malignant) and 20 healthy volunteers. LH and TGF-β serum levels were measured by ELISA. Expression of prohibitin and LHR-mRNA were assessed by IHC and TaqMan® real time gene expression assay, respectively.ResultsSerum levels of LH and TGF-β were significantly decreased among borderline and malignant groups. There was significant over-expression of LHRmRNA in malignant group. Prohibitin expression was significantly increased in malignant ovarian tissue. Strong negative correlations were found between LHR mRNA expression and serum LH levels, and between IHC score of prohibitin and serum levels of LH among patients with borderline ovarian tumors.ConclusionSteady decline of LH and TGF-B serum levels, from benign cystadenoma to borderline tumor to carcinoma, suggests their inhibitory role against OET cell growth. Increased PHB1 expression in OET suggests its proliferative activity that can be regulated by luteinisation and/or TGF-β. Furthermore increased LHR mRNA tissue expression can provide hope for using LH in treatment of some types of ovarian cancers.

Exploring the role of YAP signaling in CD4+ T cell biology

Abstract The adaptive immune system’s ability to defend against infections and destroy cancerous cells is highly dependent on T cell proliferation. A long-standing observation through in vitro and in vivo studies is that T cells cluster together during activation and subsequent proliferation. In contrast, the vast majority of other cell types turn off division machinery upon cell-cell contact. The highly conserved Hippo signaling pathway controls for cell size and number through proliferation and apoptosis, thereby making it essential for regulating organ size. At the core of the Hippo cascade is the transcription factor YAP. Upon activation, YAP translocates into the nucleus and binds with transcription enhancer factors (TEADs) to drive transcription of proliferation-related genes. While YAP signaling has been extensively studied in epithelial cells, its role in CD4+ T cell effector function and proliferation remains unexplored. Our data indicates that overall YAP protein levels rises during T cell activation. Interestingly, although YAP activation increases proliferation of epithelial cells, our data suggests that YAP is a negative regulator of cellular proliferation in CD4+ T cells. Upon YAP depletion and then restimulating T cells in vitro, we discovered that the T cells knocked down by YAP exhibited greater proliferation and produced more IL-2 than T cells transduced with control shRNA. In support of this surprising finding, expressing a constitutively active YAP with all five phosphorylation sites mutated (YAP 5SA) in T cells led to decreased proliferation of CD4+ T cells. These results hint at potentially novel functions of YAP and warrants further analysis into how YAP signaling affects T cell proliferation.

Retinoic acid has the potential to suppress endometriosis development.

BackgroundDespite endometriosis is common estrogen dependent disease afflicting women in reproductive age, the pathogenesis has not been fully elucidated. Retinoic acid has various functions in cells as biologic modulator, and aberrant retinoid metabolism seems to be involved in the lesions of endometriosis. In order to evaluate the potential of all-trans retinoic acid (ATRA) for therapeutic treatment, a transcriptome analysis and estradiol measurements in cultured endometriotic cells and tissues were conducted.MethodsThe mRNA expression levels in ATRA-treated endometriotic stromal cells (ESC) isolated from ovarian endometrial cysts (OEC) were investigated. Estradiol production in OEC tissues was also investigated.ResultsIn the isolated ESC culture supplemented with ATRA for four days, total RNA was extracted followed by a transcriptome analysis using GeneChip. Forty-nine genes were upregulated and four genes were down-regulated by the ATRA treatment. Many upregulated genes were associated with the negative regulation of cellular proliferation. In addition, ATRA treatment decreased the mRNA expression of 17-beta-dehydrogenase 2 (HSD17B2) which converts estradiol into estrone in a dose-dependent manner, and the ELISA measurements indicated that estradiol production in the OEC tissue was inhibited by ATRA treatment.ConclusionsRetinoic acid has the potential to suppress endometriosis development.

Abstract 3442: Non-canonical Wnt pathway stimulation in response to LiCl is related to suppression of cell proliferation in histologically distinct human tumors

Abstract Disturbances in cellular proliferation are a fundamental characteristic of malignancy, and have often been associated with dysregulation of the Wnt/B-catenin canonical pathway (CAN). The activity of the CAN pathway is subject to regulation by the non-canonical pathway (N-CAN) which stimulates cellular differentiation. The N-CAN pathway which has been described as a negative regulator of the CAN pathway has not been well studied in human cancers and its activity as a possible negative regulator of cellular proliferation in different tumor histologies is not known. The purpose of this study was to characterize the activation of the N-CAN pathway in relationship to tumor cell proliferation in histologically diverse human tumors. LiCl, a known activator of the CAN pathway was used to investigate effects on proliferation of different human tumors along with its capacity to regulate expression of various Wnt pathway signaling components. Cancer cells were derived from surgical specimens obtained from patients with colorectal (CRC), pancreatic (PC), renal (RCC), breast (BC), stomach (SC) and lung (LC) cancers. Cultured cells were treated with LiCl (20 mM) for 72h. Cell proliferation was determined using the MTS assay and gene expression determined using commercially available quantitative RT-PCR Wnt pathway gene expression arrays. Significant inhibition (p<0.05) of proliferation by LiCl was exhibited by all cell types except LC cells. Furthermore, suppression of proliferation was inversely correlated with upregulation of N-CAN components in all tumor types with failure to upregulate N-CAN components in LC cells. The transmembrane protein, VANGL2, which forms a receptor complex for the N-CAN pathway was upregulated significantly in 1 of 2 PC specimens and in RCC and SC cells (mean 17 fold vs. media alone). In cells with increased VANGL2 expression, PRICKLE1 (N-CAN component that interacts with VANGL2), and FRZB, an inhibitor of the CAN pathway were increased by 2.8 and 4.9 folds respectively. In these cells, additional N-CAN components such as NKD1, DAAM1, and CAN inhibitor Wif1 were increased in some, but not all histologies. In contrast, there was no effect on VANGL2 expression in CRC, 1 of 2 PC cells, and BC cells whereas upregulation of additional N-CAN genes and CAN inhibitors was observed in all tumors that demonstrated inhibition of proliferation in response to LiCl. In cancers without VANGL2 increase there was an increase in expression of the N-CAN component NKD1 (6.6 folds) and decreased expression of β-catenin (2.4 folds). Other changes in Wnt pathway genes in these cells that were observed in some but not all cell types included increased expression of FRZB and WIF1. This study demonstrates differential effects of N-CAN pathway components on the proliferation of histologically distinct human tumors. Citation Format: Donald P. Braun, Adriana Rosales, Irshad Ali. Non-canonical Wnt pathway stimulation in response to LiCl is related to suppression of cell proliferation in histologically distinct human tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3442. doi:10.1158/1538-7445.AM2014-3442

A novel variant in CDKN1C is associated with intrauterine growth restriction, short stature, and early-adulthood-onset diabetes.

CDKN1C, a cyclin-dependent kinase inhibitor and negative regulator of cellular proliferation, is paternally imprinted and has been shown to regulate β-cell proliferation. CDKN1C mutations are associated with growth disorders, including Beckwith-Wiedemann syndrome and IMAGe syndrome. To investigate the genetic basis for a familial disorder characterized by intrauterine growth restriction, short stature, and early-adulthood-onset diabetes. Genomic DNA samples (15 affected and 26 unaffected from a six-generation pedigree) were analyzed by genome-wide single nucleotide polymorphism arrays, whole exome and Sanger sequencing, and multiplex ligation-dependent probe amplification. Subjects were assessed for height, weight, adrenal gland size, ACTH, diabetes status, and testis volume. Linkage and sequence analyses were performed, and the identified genetic variant was functionally evaluated in reconstitution studies. The pedigree followed a paternally imprinted pattern of inheritance, and genetic linkage analysis identified a single significant 2.6-megabase locus on chromosome 11p15, within the imprinting center region 2. Multiplex ligation-dependent probe amplification did not detect copy number variants or methylation abnormalities. Whole exome sequencing revealed a single novel variant in the proliferating cell nuclear antigen-binding region of CDKN1C (c.842G>T, p.R281I) that co-segregated with affected status and, unlike variants found in IMAGe, did not entirely abrogate proliferating cell nuclear antigen binding. Clinical assessments revealed that affected individuals had low testicular volume but normal adrenal function. We report a novel CDKN1C mutation associated with features of IMAGe syndrome, but without adrenal insufficiency or metaphyseal dysplasia, and characterized by early-adulthood-onset diabetes. Our data expand the range of phenotypes observed with CDKN1C defects and suggest that CDKN1C mutations may represent a novel monogenic form of diabetes.

VentX trans-Activates p53 and p16ink4a to Regulate Cellular Senescence

Cell senescence is a process of irreversible arrest of cell proliferation and plays an important role in tumor suppression. Recent studies showed that Wnt inhibition is a trigger of cellular senescence. Using methods of reverse genetics, we recently identified VentX, a human homolog of the vertebrate Xenopus Vent family of homeobox genes, as a novel Wnt repressor and a putative tumor suppressor in lymphocytic leukemia. Here, we show that VentX is a direct transcriptional activator of p53-p21 and p16ink4a-Rb tumor suppression pathways. Ectopic expression of VentX in cancer cells caused an irreversible cell cycle arrest with a typical senescence-like phenotype. Conversely, inhibition of VentX expression by RNA interference ameliorated chemotherapeutic agent-induced senescence in lymphocytic leukemia cells. The results of our study further reveal the mechanisms underlying tumor suppression function of VentX and suggest a role of VentX as a potential target in cancer prevention and treatment.

Mutation of PPP2R1A: a new clue in unveiling the pathogenesis of uterine serous carcinoma

Uterine carcinoma is composed mainly of two distinct types: serous and endometrioid. Uterine serous carcinoma, while not as common as endometrioid carcinoma, is a highly aggressive disease and is associated with high mortality. Although the molecular genetic alterations of uterine endometrioid carcinoma have been well established, molecular genetic changes in uterine serous carcinoma are largely unclear. Two recent papers have analysed PPP2R1A mutations among different types of gynaecological neoplasia. Both studies report a surprisingly high frequency of somatic mutations of PPP2R1A in uterine serous carcinomas. PPP2R1A encodes a scaffolding subunit of a major serine/threonine phosphatase, protein phosphatase 2A (PP2A), which plays a critical role in diverse cellular functions, including negative regulation of cellular proliferation and potential tumour suppression. As PPP2R1A is essential for the phosphatase activity of PP2A, this observation should create a new research road map toward elucidating how PPP2R1A mutations and alterations of the PP2A pathway contribute to the pathogenesis of uterine serous carcinoma.

NM23H2 inhibits EGF- and Ras-induced proliferation of NIH3T3 cells by blocking the ERK pathway

The NM23 family proteins are involved in a variety of biological processes including tumor metastasis, development, and differentiation; however, their functions in the regulation of cellular proliferation are poorly understood. We have investigated the role of one NM23 family protein, NM23H2, in the regulation of cellular proliferation directed by the extracellular signal regulated kinase (ERK) pathway. The activity of ERKs was enhanced by knockdown of endogenous NM23H2 and blocked by overexpression of NM23H2 in both NIH3T3 and HEK293 cells. Additionally, the epidermal growth factor (EGF)- and oncogenic Ras(G12R)-induced proliferation of both HEK293 and NIH3T3 cells was reduced by NM23H2 overexpression. Furthermore, activation of Raf-1, MEK and the ERKs by either EGF or Ras(G12R) was inhibited by NM23H2 overexpression. Together, our data indicate that NM23H2 is a negative regulator of cellular proliferation stimulated by EGF- and Ras-mediated activation of the ERK pathway.

Proteomic Analysis of the Role of STAT5A in the Apoptosis of Human Pre-B Cells : Evidence for a Connection to Daxx and the Oxidative Stress

It is well established that STAT5A and 5B transcription factors (TF) play a major role in hematopoiseis and oncogenesis. In particular, there is a body of evidence suggesting that they might be involved in B lymphocyte survival, proliferation and development as well as in B cell neoplastic transformation. To investigate the role of STAT5A in human precursor B cell survival, we stably transfected a dominant negative form of STAT5A (DN-STAT5A, deleted in its transactivation domain) in the human leukemic Nalm6 pre-B cell line. All clones expressing DN-STAT5A exhibited a lower proliferation rate associated with a higher spontaneous apoptosis and that was massively enhanced upon IL7 stimulation. They also were more sensitive to FAS (CD95)- and at a lesser extent to etoposide-induced cell death than cells transfected with the empty vector (Nalm6neo), suggesting a hitherto unknown link between STAT5A and apoptosis/survival pathways. There was no evidence for changes in the levels of expression of Bcl2, BclxL, Bad or –c-Myc in DN-STAT5A expressing cells whereas Bax and Bim expression was sharply up-regulated in these cells. In order to identify the proteins involved in the connection between Fas and STAT5A, we used a proteomic approach with differential analysis of cells expressing (Nalm6Δ5A749) or not (Nalm6neo) the DN-STAT5A. Statistical analysis of bidimensional (2D) gels and use of MALDI-TOF technique enabled us to identify 7 proteins down-regulated in the Nalm6Δ5A749 cells, including members of heat shock proteins such as hsp27 and hsp70, as well as proteins implied in the control of oxidative stress like glutathione synthetase and transaldolase. By contrast, whereas 6 proteins were shown to be up-regulated, including prohibitin which is a negative regulator of cellular proliferation. Nalm6Δ5A749 cells exhibited enhanced levels of reactive oxygen species in keeping with the idea that oxidative stress might be involved in the increased sensitivity of these cells to apoptotic signals. Although expression of all above mentioned molecules did not change upon Fas stimulation, addition of gluthatione (10mM) resulted in a complete inhibition of Fas-mediated apoptosis in Nalm6Δ5A749 cells. Whether or not prohibitin might be involved in the decreased proliferation rate in DN-STAT5A expressing cells needs further investigation. As Hsp27 has been previously shown to have the potential to sequestrate Daxx, a molecule that is involved in Fas receptor signaling, we investigated the subcellular location of this molecule in Nalm6Δ5A749 cells. We showed that Daxx was essentially expressed in the cytoplasm of these cells whereas it was mainly located in the nucleus in Nalm6neo cells. Our results bring evidence for a role of STAT5A in pre-B cell growth and survival and in particular for a connection between this TF and Fas signaling machinery that might involve Hsp27 and Daxx. They also point out to a so far undescribed link between STAT5 and oxidative stress.

The type III TGF- receptor signals through both Smad3 and the p38 MAP kinase pathways to contribute to inhibition of cell proliferation

Transforming growth factor beta (TGFbeta) has an important role as a negative regulator of cellular proliferation. The type III transforming growth factor beta receptor (TbetaRIII) has an emerging role as both a TGFbeta superfamily co-receptor and in mediating signaling through its cytoplasmic domain. In L6 myoblasts, TbetaRIII expression enhanced TGFbeta1-mediated growth inhibition, with this effect mediated, in part, by the TbetaRIII cytoplasmic domain. The effects of TbetaRIII were not due to altered ligand presentation or to differences in Smad2 phosphorylation. Instead, TbetaRIII specifically increased Smad3 phosphorylation, both basal and TGFbeta-stimulated Smad3 nuclear localization and Smad3-dependent activation of reporter genes independent of its cytoplasmic domain. Conversely, SB431542, a type I transforming growth factor beta receptor (TbetaRI) inhibitor, as well as dominant-negative Smad3 specifically and significantly abrogated the effects of TbetaRIII on TGFbeta1-mediated inhibition of proliferation. TbetaRIII also specifically increased p38 phosphorylation, and SB203580, a p38 kinase inhibitor, specifically and significantly abrogated the effects of TbetaRIII/TGFbeta1-mediated inhibition of proliferation in L6 myoblasts and in primary human epithelial cells. Importantly, treatment with the TbetaRI and p38 inhibitors together had additive effects on abrogating TbetaRIII/TGFbeta1-mediated inhibition of proliferation. In a reciprocal manner, short hairpin RNA-mediated knockdown of endogenous TbetaRIII in various human epithelial cells attenuated TGFbeta1-mediated inhibition of proliferation. Taken together, these data demonstrate that TbetaRIII contributes to and enhances TGFbeta-mediated growth inhibition through both TbetaRI/Smad3-dependent and p38 mitogen-activated protein kinase pathways.

Roles of heme oxygenase-1 in curcumin-induced growth inhibition in rat smooth muscle cells

In vascular smooth muscle cells (VSMCs), induction of the heme oxygenase-1 (HO-1) confers vascular protection against cellular proliferation mainly via its up-regulation of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) that is involved in negative regulation of cellular proliferation. In the present study, we investigated whether the phytochemical curcumin and its metabolite tetrahydrocurcumin could induce HO-1 expression and growth inhibition in rat VSMCs and, if so, whether their antiproliferative effect could be mediated via HO-1 expression. At non-toxic concentrations, curcumin possessing two Michael-reaction acceptors induced HO-1 expression by activating antioxidant response element (ARE) through translocation of the nuclear transcription factor E2-related factor-2 (Nrf2) into the nucleus and also inhibited VSMC growth triggered by 5% FBS in a dose-dependent manner. In contrast, tetrahydrocurcumin lacking Michael-reaction acceptor showed no effect on HO-1 expression, ARE activation and VSMC growth inhibition. The antiproliferative effect of curcumin in VSMCs was accompanied by the increased expression of p21(WAF1/CIP1). Inhibition of VSMC growth and expression of p21(WAF1/CIP1) by curcumin were partially, but not completely, abolished when the cells were co- incubated with the HO inhibitor tin protoporphyrin. In human aortic smooth muscle cells (HASMCs), curcumin also inhibited growth triggered by TNF-alpha and increased p21(WAF1/CIP1) expression via HO-1-dependent manner. Our findings suggest that curcumin has an ability to induce HO-1 expression, presumably through Nrf2-dependent ARE activation, in rat VSMCs and HASMCs, and provide evidence that the antiproliferative effect of curcumin is considerably linked to its ability to induce HO-1 expression.

Caveolin-1 sensitizes rat pituitary adenoma GH3 cells to bromocriptine induced apoptosis

BackgroundProlactinoma is the most frequent pituitary tumor in humans. The dopamine D2 receptor agonist bromocriptine has been widely used clinically to treat human breast tumor and prolactinoma through inhibition of hyperprolactinemia and induction of tumor cell apoptosis, respectively, but the molecular mechanism of bromocriptine induction of pituitary tumor apoptosis remains unclear. Caveolin-1 is a membrane-anchored protein enriched on caveolae, inverted flask-shaped invaginations on plasma membranes where signal transduction molecules are concentrated. Currently, caveolin-1 is thought to be a negative regulator of cellular proliferation and an enhancer of apoptosis by blocking signal transduction between cell surface membrane receptors and intracellular signaling protein cascades. Rat pituitary adenoma GH3 cells, which express endogenous caveolin-1, exhibit increased apoptosis and shrinkage after exposure to bromocriptine. Hence, the GH3 cell line is an ideal model for studying the molecular action of bromocriptine on prolactinoma.ResultsThe expression of endogenous caveolin-1 in GH3 cells was elevated after bromocriptine treatment. Transiently expressed mouse recombinant caveolin-1 induced apoptosis in GH3 cells by enhancing the activity of caspase 8. Significantly, caveolin-1 induction of GH3 cell apoptosis was sensitized by the administration of bromocriptine. Phosphorylation of caveolin-1 at tyrosine 14 was enhanced after bromocriptine treatment, suggesting that bromocriptine-induced phosphorylation of caveolin-1 may contribute to sensitization of apoptosis in GH3 cells exposed to bromocriptine.ConclusionOur results reveal that caveolin-1 increases sensitivity for apoptosis induction in pituitary adenoma GH3 cells and may contribute to tumor shrinkage after clinical bromocriptine treatment.

Epigenetic alterations of BRG1 leads to cancer development through its nuclear-cytoplasmic shuttling abnormalities

SWI/SNF is a multiprotein chromatin remodeling complex important for gene regulation. BRG1 and its close relative BRM, have ATPase activity necessary for transcriptional regulation by conformational change of nucleosomes. Due to this role on gene expression, several members of SWI/SNF complex including BRG1 and BRM function as a tumor suppressor or negative regulator of cellular proliferation. On the other hand, the shuttling of proteins between nucleus and cytoplasm is strongly involved in the regulation of cell cycle and proliferation. Many of tumor suppressor gene (TSG)s including p53, BRCA1, ING1 play some of their functions through nucleocytoplasmic shuttling. Abnormalities related with this process abrogate the subcellular localization of the TSGs and lead to cancer development. We recently demonstrated BRG1 as a TSG in oral cancer. Our analysis also revealed an interesting finding that one of the splicing forms of BRG1 is selectively lost in cancer tissue as compared to normal counterparts. Our further analysis revealed a putative nuclear retention signal domain for this splicing form. In this article, we speculate the possible mechanism for the inactivation of BRG1 gene in oral cancer through an abnormality in its subcellular localization.

RB reversibly inhibits DNA replication via two temporally distinct mechanisms.

The retinoblastoma (RB) tumor suppressor is a critical negative regulator of cellular proliferation. Repression of E2F-dependent transcription has been implicated as the mechanism through which RB inhibits cell cycle progression. However, recent data have suggested that the direct interaction of RB with replication factors or sites of DNA synthesis may contribute to its ability to inhibit S phase. Here we show that RB does not exert a cis-acting effect on DNA replication. Furthermore, the localization of RB was distinct from replication foci in proliferating cells. While RB activation strongly attenuated the RNA levels of multiple replication factors, their protein expression was not diminished coincident with cell cycle arrest. During the first 24 h of RB activation, components of the prereplication complex, initiation factors, and the clamp loader complex (replication factor C) remained tethered to chromatin. In contrast, the association of PCNA and downstream components of the processive replication machinery was specifically disrupted. This signaling from RB occurred in a manner dependent on E2F-mediated transcriptional repression. Following long-term activation of RB, we observed the attenuation of multiple replication factors, the complete cessation of DNA synthesis, and impaired replicative capacity in vitro. Therefore, functional distinctions exist between the "chronic" RB-mediated arrest state and the "acute" arrest state. Strikingly, attenuation of RB activity reversed both acute and chronic replication blocks. Thus, continued RB action is required for the maintenance of two kinetically and functionally distinct modes of replication inhibition.

The Regulatory Expression of Procollagen COOH-Terminal Proteinase Enhancer in the Proliferation of Vascular Smooth Muscle Cells

Intimal hyperplasia following arterial endothelial denudation results in large part from the proliferation of vascular smooth muscle cells (SMCs) and matrix accumulation. Procollagen COOH-terminal proteinase enhancer (PCPE) binds procollagen COOH-propeptides and potentiates procollagen COOH-proteinase activity to cleave COOH-propeptides of procollagens I–III. Here we report the enhanced expression of PCPE in cultured SMCs and in intimal thickening induced by arterial injury. The levels of PCPE mRNA in parallel with the level of p21Cip1 mRNA, as a negative regulator of cellular proliferation, increased under serum deprivation or reduced cellular proliferation in cultured SMCs. In contrast, rapidly proliferating cells show the decreased levels of PCPE mRNA. In vivo, the marked induction of PCPE in injured rat arteries occurred at 14 days after endothelial denudation. The induced expression levels of PCPE as well as p21Cip1 were maintained until 42 days, although cyclin E expression declined. Furthermore, transforming growth factor β1 (TGF-β1), an important regulator of cellular proliferation in atheroma, increased the levels of the PCPE mRNA in cultured SMCs. Thus, the regulatory expression of PCPE dependent on cellular proliferation, and particularly contact inhibition, may play a key role in the proliferation of SMCs and matrix production during the process of atheroma formation.

P300 Protein as a Coactivator of GATA-5 in the Transcription of Cardiac-restricted Atrial Natriuretic Factor Gene

A cellular target of adenovirus E1A oncoprotein, p300 is a transcriptional coactivator and a negative regulator of cellular proliferation. A previous study suggests that the p300 family is also involved in cell type-specific transcription in cardiac myocytes. However, nothing is known about which cardiac transcription factor(s) interact with and transactivate through these proteins. The transcription factors GATA-4/5/6 have been implicated as key regulators of cardiogenesis, and they participate in the transcription of many cardiac-specific genes. Here we show that E1A represses the GATA-5-dependent transactivation of a promoter derived from the cardiac-restricted atrial natriuretic factor gene. This repression is correlated with the interaction of E1A with p300, indicating that p300 participates in GATA-5-dependent transactivation. E1A markedly down-regulates endogenous atrial natriuretic factor expression, as well as disrupts the interaction between p300 and GATA-5. A small fragment of p300 containing the carboxyl-terminal cysteine/histidine-rich domain, sufficient to interact with GATA-5, prevents transcriptional activation by GATA-5 as a dominant-negative mutant. Consistent with its role as a coactivator, p300 markedly potentiates GATA-5-activated transcription. These results implicate p300 as an important component of myocardial cell differentiation and provide an insight into the relationship between mechanisms that mediate cell type-specific transcription and cell cycle regulation during cardiogenesis.

Viral Ski Inhibits Retinoblastoma Protein (Rb)-mediated Transcriptional Repression in a Dominant Negative Fashion

The mechanism by which the viral oncogene ski (v-ski) transforms chicken embryo fibroblasts is currently unknown. Recently, the c-ski gene product (c-Ski) was found to bind to N-CoR (nuclear hormone receptor co-repressor), an element implicated in transcriptional repression mediated by multiple transcriptional repressors including the nuclear hormone receptors and Mad. c-Ski is required for transcriptional repression mediated by Mad involved in negative regulation of cellular proliferation. v-Ski abrogates Mad-induced transcriptional repression in a dominant negative fashion. Here we report that v-Ski also inhibits transcriptional repression mediated by Rb, another tumor suppressor gene product. Rb forms a complex with c-Ski, Sin3A, and histone deacetylase (HDAC) via direct binding to c-Ski and HDAC. c-Ski is required for the transcriptional repression mediated by Rb. These results suggest that inhibition of Rb activity contributes, at least partly, to transformation by v-Ski.

Heat shock proteins in middle ear cholesteatoma

Heat shock proteins (HSPs), particularly HSP60 and HSP70, may have integral roles in several aspects of the pathogenesis of acquired cholesteatoma. Cholesteatoma is usually associated with an inflammatory reaction occurring in the middle ear cavity, leading to proliferation, differentiation, and programmed cell death of keratinocytes. The presence of HSP60 and HSP70 in cholesteatoma was demonstrated by the immunoblotting assay with specific anti-HSP60 and anti-HSP70 antibodies after protein extraction. The distribution of HSP60 and HSP70 in the cholesteatoma tissues was studied immunohistochemically with paraffin-embedded sections. HSP60 and HSP70 appeared to be localized in the cytoplasm of keratinocytes in all layers of the epithelium of cholesteatoma. Moreover, HSP70 was found in the nucleus of keratinocytes. Both HSPs were not labeled in the epidermis of the external ear canal skin or normal facial skin, except for significant staining on keratinocytes of hair follicles in facial skin. High density of HSP60 and HSP70 may be induced by the inflammatory reaction and immune responses in the middle ear cavity during the clinical development of cholesteatoma. Induction of HSP60 and HSP70 in cholesteatoma epithelium may also be related to hyperproliferation and active differentiation of basal keratinocytes, which cause the accumulation of keratin debris, a characteristic feature of cholesteatoma. In the nucleus of keratinocytes, HSP70 may act to stabilize p53 protein, which functions as a negative regulator of cellular proliferation and is crucial to apoptosis of keratinocytes. This study demonstrates HSPs in cholesteatoma and suggests important roles for HSPs in the clinical development of cholesteatoma. (Otolaryngol Head Neck Surg 1996;114:77-83.)

Heat shock proteins in middle ear cholesteatoma.

Heat shock proteins (HSPs), particularly HSP60 and HSP70, may have integral roles in several aspects of the pathogenesis of acquired cholesteatoma. Cholesteatoma is usually associated with an inflammatory reaction occurring in the middle ear cavity, leading to proliferation, differentiation, and programmed cell death of keratinocytes. The presence of HSP60 and HSP70 in cholesteatoma was demonstrated by the immunoblotting assay with specific anti-HSP60 and anti-HSP70 antibodies after protein extraction. The distribution of HSP60 and HSP70 in the cholesteatoma tissues was studied immunohistochemically with paraffin-embedded sections. HSP60 and HSP70 appeared to be localized in the cytoplasm of keratinocytes in all layers of the epithelium of cholesteatoma. Moreover, HSP70 was found in the nucleus of keratinocytes. Both HSPs were not labeled in the epidermis of the external ear canal skin or normal facial skin, except for significant staining on keratinocytes of hair follicles in facial skin. High density of HSP60 and HSP70 may be induced by the inflammatory reaction and immune responses in the middle ear cavity during the clinical development of cholesteatoma. Induction of HSP60 and HSP70 in cholesteatoma epithelium may also be related to hyperproliferation and active differentiation of basal keratinocytes, which cause the accumulation of keratin debris, a characteristic feature of cholesteatoma. In the nucleus of keratinocytes, HSP70 may act to stabilize p53 protein, which functions as a negative regulator of cellular proliferation and is crucial to apoptosis of keratinocytes. This study demonstrates HSPs in cholesteatoma and suggests important roles for HSPs in the clinical development of cholesteatoma.