The motility of glioblastoma tumour cells is modulated by intracellular cofilin expression in a concentration-dependent manner

The invasive behaviour of tumour cells has been attributed in part to dysregulated cell motility. Members of the ADF/Cofilin family of actin-binding proteins are known to increase microfilament dynamics by increasing the rate at which actin monomers leave the pointed end of the filament and by a filament-severing activity. As depolymerisation is a rate-limiting step in actin dynamics, ADF/Cofilins are suspected to facilitate the motility of cells. To test this, we investigated the influence of cofilin on tumour motility by transient and stably overexpressing cofilin in the human glioblastoma cell line, U373 MG. Several different methods were used to ascertain the level of cofilin in overexpressing clones and this was correlated with their rate of random locomotion. A biphasic relationship between cofilin level and locomotory rate was found. Clones that displayed a moderate amount of overproduction of cofilin were found to have increased rates of locomotion approximately linear to the overproduction of cofilin up to an optimal cofilin level of about 4.5 times that of wild type cells at which the cells were almost twice as fast. However, clones producing more than this optimal amount were found to locomote at progressively reduced speeds. Cells that overexpress cofilin have reduced stress fibres compared to control cells showing that the excess cofilin affects the actin cytoskeleton. We conclude that overexpression of cofilin enhances the motility of glioblastoma tumour cells in a concentration-dependent fashion, which is likely to contribute to their invasiveness.     

SVEP1 expression is regulated in estrogen-dependent manner

The SVEP1 protein comprises modules related to the selectin super family and other motifs found in cell surface molecules. Earlier, we demonstrated that SVEP1 is expressed in osteogenic cells both in vivo and in vitro; in the current study we elaborate on the regulation of SVEP1 by 17beta-Estradiol (17betaE2). SVEP1 message is expressed in vivo by bone marrow cells of sham-operated rats, but not in estrogen-depleted ovariectomized (OVX) rats. We demonstrated that 17betaE2 treatment increases the level of the SVEP1 expression in cultured osteoblasts. SVEP1 was identified also in breast carcinoma (BC) cells known to reside in bone when metastasized from the primary tumor. SVEP1 expression was demonstrated by immunohistochemistry and fluorescence-activated cell sorting (FACS) on various BC cell lines. The chromatin immunoprecipitation (ChIP) assay was applied to analyze the estrogen receptor (ER) binding to the putative SVEP1 promoter. We demonstrated that treatment with 17betaE2 or ICI 182,780 affects this binding and regulates the mRNA and protein levels of SVEP1 in BC cells. We propose that SVEP1 may serve as a useful biomarker for studying the mechanism of cells interactions within the local microenvironment affected by estrogen.     

The expression of tumstatin is down-regulated in renal carcinoma

Tumstatin is the 28 kDa NC1 domain of the α3 chain of type IV collagen that inhibits pathological angiogenesis and suppresses endothelial cell proliferation and tumor growth. In the present paper, we expressed and purified recombinant human tumstatin protein and then prepared the anti-tumstatin polyclonal antibody. To investigate the expression of tumstatin in renal carcinoma, tumstatin protein was detected by western blotting using the prepared anti-tumstatin antibody and tumstatin mRNA levels were assayed by RT-PCR. The results showed that the expression of tumstatin gene was down-regulated in renal carcinoma tissues and cells. Our study suggests that as a novel endogenous angiogenesis inhibitor, tumstatin gene expression may be a marker for diagnosis, therapy and prognosis of renal carcinoma.     

Niemann-Pick C1 like 1 gene expression is down-regulated by LXR activators in the intestine

Niemann-Pick C1 like 1 (NPC1L1) is a protein critical for intestinal cholesterol absorption. The nuclear receptors peroxisome proliferator-activated receptor alpha (PPARalpha) and liver X receptors (LXRalpha and LXRbeta) are major regulators of cholesterol homeostasis and their activation results in a reduced absorption of intestinal cholesterol. The goal of this study was to define the role of PPARalpha and LXR nuclear receptors in the regulation of NPC1L1 gene expression. We show that LXR activators down-regulate NPC1L1 mRNA levels in the human enterocyte cell line Caco-2/TC7, whereas PPARalpha ligands have no effect. Furthermore, NPC1L1 mRNA levels are decreased in vivo, in duodenum of mice treated with the LXR agonist T0901317. In conclusion, the present study identifies NPC1L1 as a novel LXR target gene further supporting a crucial role of LXR in intestinal cholesterol homeostasis.     

Syndecan-1 expression is regulated in an isoform-specific manner by the farnesoid-X receptor

Syndecan-1 (SDC1), a transmembrane heparan sulfate proteoglycan that participates in the binding and internalization of extracellular ligands, was identified in a screen designed to isolate genes that are regulated by the farnesoid X-receptor (FXR, NR1H4). Treatment of human hepatocytes with either naturally occurring (chenodeoxycholic acid) or synthetic (GW4064) FXR ligands resulted in both induction of SDC1 mRNA and enhanced binding, internalization, and degradation of low density lipoprotein. Transient transfection assays, using wild-type and mutant SDC1 promoter-luciferase genes, led to the identification of a nuclear hormone receptor-binding hexad arranged as a direct repeat separated by one nucleotide (DR-1) in the proximal promoter that was necessary and sufficient for activation by FXR. The wild-type, but not a mutated DR-1 element, conferred FXR responsiveness to a heterologous thymidine kinase promoter-reporter gene. Four murine FXR isoforms have been identified recently that differ either at their amino terminus and/or by the presence or absence of four amino acids in the hinge region. Interestingly, the activities of the human SDC1 promoter-reporter constructs were highly induced by the two FXR isoforms that do not contain the four-amino acid insert and were unresponsive to the isoforms containing the four amino acids. Thus, current studies demonstrate that hepatic SDC1 is induced in an FXR isoform-specific manner. Increased expression of SDC1 may account in part for the hypotriglyceridemic effect that can result from the administration of chenodeoxycholic acid to humans.     

Syndecan-1 expression in epithelial cells is induced by transforming growth factor beta through a PKA-dependent pathway

Syndecans comprise a major family of cell surface heparan sulfate proteoglycans (HSPGs). Syndecans bind and modulate a wide variety of biological molecules through their heparan sulfate (HS) moiety. Although all syndecans contain the ligand binding HS chains, they likely perform specific functions in vivo because their temporal and spatial expression patterns are different. However, how syndecan expression is regulated has yet to be clearly defined. In this study, we examined how syndecan-1 expression is regulated in epithelial cells. Our results showed that among several bioactive agents tested, only forskolin and three isoforms of TGFbeta (TGFbeta1-TGFbeta3) significantly induced syndecan-1, but not syndecan-4, expression on various epithelial cells. Steady-state syndecan-1 mRNA was not increased by TGFbeta treatment and cycloheximide did not inhibit syndecan-1 induction by TGFbeta, indicating that TGFbeta induces syndecan-1 in a post-translational manner. However, TGFbeta induction of syndecan-1 was inhibited by transient expression of a dominant-negative construct of protein kinase A (PKA) and by specific inhibitors of PKA. Further (i) syndecan-1 cytoplasmic domains were Ser-phosphorylated when cells were treated with TGFbeta and this was inhibited by a PKA inhibitor, (ii) PKA was co-immunoprecipitated from cell lysates by anti-syndecan-1 antibodies, (iii) PKA phosphorylated recombinant syndecan-1 cytoplasmic domains in vitro, and (iv) expression of a syndecan-1 construct with its invariant Ser(286) replaced with a Gly was not induced by TGFbeta. Together, these findings define a regulatory mechanism where TGFbeta signals through PKA to phosphorylate the syndecan-1 cytoplasmic domain and increases syndecan-1 expression on epithelial cells.     

ALAS1 gene expression is down-regulated by Akt-mediated phosphorylation and nuclear exclusion of FOXO1 by vanadate in diabetic mice

Porphyrias are diseases caused by partial deficiencies of haem biosynthesis enzymes. Acute porphyrias are characterized by a neuropsychiatric syndrome with intermittent induction of hepatic ALAS1 (δ-aminolaevulinate synthase 1), the first and rate-limiting enzyme of the haem pathway. Acute porphyria attacks are usually treated by the administration of glucose; its effect is apparently related to its ability to inhibit ALAS1 by modulating insulin plasma levels. It has been shown that insulin blunts hepatocyte ALAS1 induction, by disrupting the interaction of FOXO1 (forkhead box O1) and PGC-1α (peroxisome-proliferator-activated receptor γ co-activator 1α). We evaluated the expression of ALAS1 in a murine model of diabetes and determined the effects of the insulinomimetic vanadate on the enzyme regulation to evaluate its potential for the treatment of acute porphyria attacks. Both ALAS1 mRNA and protein content were induced in diabetic animals, accompanied by decreased Akt phosphorylation and increased nuclear FOXO1, PGC-1α and FOXO1-PGC-1α complex levels. Vanadate reversed ALAS1 induction, with a concomitant PI3K (phosphoinositide 3-kinase)/Akt pathway activation and subsequent reduction of nuclear FOXO1, PGC-1α and FOXO1-PGC-1α complex levels. These findings support the notion that the FOXO1-PGC-1α complex is involved in the control of ALAS1 expression and suggest further that a vanadate-based therapy could be beneficial for the treatment of acute porphyria attacks.     

Androgen receptor expression in the rat prostate is down-regulated by dietary phytoestrogens

Background  

It is well established that the growth of the prostate gland is a hormone-dependent phenomenon involving both androgenic and estrogenic control. Proliferation of prostate cells is, at least in part, under control of estrogen receptor beta (ER-beta). Phytoestrogens bind ER-beta with high affinity and therefore may have antiproliferative effects in the prostate.     

Inhibition of c-Jun N-terminal kinase 2 expression suppresses growth and induces apoptosis of human tumor cells in a p53-dependent manner

c-Jun N-terminal kinase (JNK) plays a critical role in coordinating the cellular response to stress and has been implicated in regulating cell growth and transformation. To investigate the growth-regulatory functions of JNK1 and JNK2, we used specific antisense oligonucleotides (AS) to inhibit their expression. A survey of several human tumor cell lines revealed that JNKAS treatment markedly inhibited the growth of cells with mutant p53 status but not that of cells with normal p53 function. To further examine the influence of p53 on cell sensitivity to JNKAS treatment, we compared the responsiveness of RKO, MCF-7, and HCT116 cells with normal p53 function to that of RKO E6, MCF-7 E6, and HCT116 p53(-/-), which were rendered p53 deficient by different methods. Inhibition of JNK2 (and to a lesser extent JNK1) expression dramatically reduced the growth of p53-deficient cells but not that of their normal counterparts. JNK2AS-induced growth inhibition was correlated with significant apoptosis. JNK2AS treatment induced the expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) in parental MCF-7, RKO, and HCT116 cells but not in the p53-deficient derivatives. That p21(Cip1/Waf1) expression contributes to the survival of JNK2AS-treated cells was supported by additional experiments demonstrating that p21(Cip1/Waf1) deficiency in HCT116 cells also results in heightened sensitivity to JNKAS treatment. Our results indicate that perturbation of JNK2 expression adversely affects the growth of otherwise nonstressed cells. p53 and its downstream effector p21(Cip1/Waf1) are important in counteracting these detrimental effects and promoting cell survival.     

Keratin 13 expression is linked to squamous differentiation in rabbit tracheal epithelial cells and down-regulated by retinoic acid

Rabbit tracheal epithelial (RbTE) cells in primary culture undergo at confluence a multistep program of squamous differentiation. This study examines the expression of keratins in RbTE cells in relation to this differentiation process. During the exponential growth phase RbTE cells are undifferentiated and express three major keratins, K5, K14, and K19, and two minor keratins, K6 and K16. Squamous differentiation is accompanied by increased expression of keratins K6, K16, and K19, and in particular of keratin K13, which reacts specifically with the monoclonal antibody AE8. These changes in keratin synthesis coincide with the commitment to terminal differentiation. Retinoic acid, an inhibitor of the expression of the squamous differentiated phenotype, inhibits the increase in the expression of K6, K16, and K13 and reduces the expression of K5 and K14; however, retinoic acid treatment results in increased levels of keratin K19 and K18. Retinoic acid inhibits the expression of K16 and K13 at concentrations as low as 10(-9)-10(-10) M. At least some of these changes in keratins appear to be related to alterations in the cellular levels of the respective mRNAs. Our results indicate that specific changes in keratin expression, in particular keratin K13, correlate with the onset of squamous differentiation in RbTE cells. Induction of the expression of keratin K13 may function as a marker of squamous differentiation in tracheobronchial epithelial cells.     

CD44 and TGFbeta1 synergise to induce expression of a functional NADPH oxidase in promyelocytic cells

Bone marrow stromal cells produce large amounts of extracellular matrix and cytokines. Amongst them, hyaluronan, a glycosaminoglycan and ligand for the cell surface molecule CD44, and TGFbeta1, a cytokine particularly important in monocyte differentiation. We have studied in vitro the role of hyaluronan and TGFbeta1 in the differentiation process of U937 monocytic progenitor cells. We provide evidence that, in the presence of whole blood-derived serum, the addition of hyaluronan is sufficient to induce the expression of NADPH-oxidase components but not of other monocytic markers (CD14, CD11b, and VLA-4). In the presence of plasma-derived serum, besides hyaluronan, the additional presence of TGFbeta1 was required for the expression of all of the components of the NADPH oxidase. We further show that hyaluronan mediates its effect through CD44. We conclude that cell matrix factors act cooperatively with cytokines to induce the expression of the components of the NADPH-oxidase in monocytic progenitor cells.