Actin-Mediated Gene Expression Depends on RhoA and Rac1 Signaling in Proximal Tubular Epithelial Cells

Morphological alterations of cells can lead to modulation of gene expression. An essential link is the MKL1-dependent activation of serum response factor ( SRF), which translates changes in the ratio of G-and F-actin into mRNA transcription. SRF activation is only partially characterized in non-transformed epithelial cells. Therefore, the impact of GTPases of the Rho family and changes in F-actin structures were analyzed in renal proximal tubular epithelial cells. Activation of SRF signaling was compared to the regulation of a known MKL1/ SRF target gene, connective tissue growth factor ( CTGF). In the human proximal tubular cell line HKC-8 overexpression of two actin mutants either favoring or preventing the formation of F-actin fibers regulated SRF-mediated transcription as well as CTGF expression. Only overexpression of constitutively active RhoA activated SRF-dependent gene expression whereas no effect was detected upon overexpression of Rac1 mutants. To elucidate the functional role of Rho kinases as downstream mediators of RhoA, pharmacological inhibition and genetic inhibition by transient siRNA knock down were compared. Upon stimulation with lysophosphatidic acid ( LPA) Rho kinase inhibitors partially suppressed SRF-mediated transcription, whereas interference with Rho kinase expression by siRNA reduced activation of SRF, but barely affected CTGF expression. Together with the partial inhibition of CTGF expression by the pharmacological inhibitors Y27432 and H1154, Rho kinases seem to be less important in mediating RhoA signaling related to CTGF expression in HKC8 epithelial cells. Short term pharmacological inhibition of Rac1 activity by EHT1864 reduced SRF-dependent CTGF expression in HKC-8 cells, but was overcome by a stimulatory effect after prolonged incubation after 4-6 h. Similarly, human primary cells of proximal but not of distal tubular origin showed inhibitory as well as stimulatory effects of Rac1 inhibition. Thus, RhoA signaling activates MKL1-SRF-mediated CTGF expression in proximal tubular cells, whereas Rac1 signaling is more complex with adaptive cellular responses

Lack of α8 integrin leads to morphological changes in renal mesangial cells, but not in vascular smooth muscle cells

<p>Abstract</p> <p>Background</p> <p>Extracellular matrix receptors of the integrin family are known to regulate cell adhesion, shape and functions. The α8 integrin chain is expressed in glomerular mesangial cells and in vascular smooth muscle cells. Mice deficient for α8 integrin have structural alterations in glomeruli but not in renal arteries. For this reason we hypothesized that mesangial cells and vascular smooth muscle cells differ in their respective capacity to compensate for the lack of α8 integrin.</p> <p>Results</p> <p>Wild type and α8 integrin-deficient mesangial cells varied markedly in cell morphology and expression or localization of cytoskeletal molecules. In α8 integrin-deficient mesangial cells α-smooth muscle actin and CTGF were downregulated. In contrast, there were no comparable differences between α8 integrin-deficient and wild type vascular smooth muscle cells. Expression patterns of integrins were altered in α8 integrin-deficient mesangial cells compared to wild type mesangial cells, displaying a prominent overexpression of α2 and α6 integrins, while expression patterns of the these integrins were not different between wild type and α8 integrin-deficient vascular smooth muscle cells, respectively. Cell proliferation was augmented in α8 integrin-deficient mesangial cells, but not in vascular smooth muscle cells, compared to wild type cells.</p> <p>Conclusions</p> <p>Our findings suggest that α8 integrin deficiency has differential effects in mesangial cells and vascular smooth muscle cells. While the phenotype of vascular smooth muscle cells lacking α8 integrin is not altered, mesangial cells lacking α8 integrin differ considerably from wild type mesangial cells which might be a consequence of compensatory changes in the expression patterns of other integrins. This could result in glomerular changes in α8 integrin-deficient mice, while the vasculature is not affected in these mice.</p

Non-professional phagocytosis: a general feature of normal tissue cells

Non-professional phagocytosis by cancer cells has been described for decades. Recently, non-professional phagocytosis by normal tissue cells has been reported, which prompted us to take a closer look at this phenomenon. Non-professional phagocytosis was studied by staining cultured cells with live-cell staining dyes or by staining paraffin-embedded tissues by immunohistochemistry. Here, we report that each of 21 normal tissue cell lines from seven different organs was capable of phagocytosis, including ex vivo cell cultures examined before the 3rd passage as well as the primary and virus-transformed cell lines. We extended our analysis to an in vivo setting, and we found the occurrence of non-professional phagocytosis in healthy skin biopsies immediately after resection. Using dystrophin immunohistochemistry for membrane staining, human post-infarction myocardial tissue was assessed. We found prominent signs of non-professional phagocytosis at the transition zone of healthy and infarcted myocardia. Taken together, our findings suggest that non-professional phagocytosis is a general feature of normal tissue cells

Cell type-specific regulation of CCN2 protein expression by PI3K–AKT–FoxO signaling

The biological activity of connective tissue growth factor (CTGF, CCN2) is regulated at the level of intracellular signaling leading to gene expression, and by its extracellular interaction partners which determine the functional outcome of CCN2 action. In this overview, we summarize the data which provide evidence that one of the major signaling pathways, phosphatidylinositol-3 kinase (PI3K)–AKT signaling, shows a remarkable cell type-dependence in terms of regulation of CCN2 expression. In smooth muscle cells, fibroblasts, and epithelial cells, inhibition of this pathway either reduced CCN2 expression or was not involved in CCN2 gene expression depending on the stimulus used. In microvascular endothelial cells by contrast, activation of PI3K–AKT signaling was inversely related to CCN2 expression. Upregulation of CCN2 upon inhibition of PI3K–AKT was also observed in primary cultures of human endothelial cells (HUVEC) exposed to laminar flow in an in vitro flow-through system. In different types of endothelial cells, FoxO transcription factors, which are negatively regulated by AKT, were identified as potent activators of CCN2 gene expression. In HUVEC, we observed a correlation between enhanced nuclear localization of FoxO1 and increased synthesis of CCN2 protein in areas of non-uniform shear stress. These data indicate that FoxO proteins are key regulators of CCN2 gene expression which determine the effect of PI3K–AKT activation in terms of CCN2 regulation. Short summary Phosphatidylinositol-3 kinase (PI3K)–AKT signaling shows a remarkable cell type-dependence in terms of regulation of CCN2 expression. In endothelial cells activation of PI3K - AKT signaling was inversely related to CCN2 expression. FoxO transcription factors, which are negatively regulated by AKT, were identified as potent activators of CCN2 gene expression

Simvastatin and the Rho-kinase inhibitor Y-27632 prevent myofibroblast transformation in Peyronie's disease-derived fibroblasts via inhibition of YAP/TAZ nuclear translocation.

OBJECTIVES To uncover the anti-myofibroblast (MFB) properties of Rho-kinase inhibitor (compound Y-27632) and simvastatin in an in vitro model of Peyronie's disease (PD), a sexually debilitating disease caused by an irreversible fibrotic plaque in the penile tunica albuginea (TA). MATERIALS AND METHODS Primary human fibroblasts (FBs) were isolated from surgically obtained TA tissue from patients with PD. To induce MFB status, cells were stimulated with 3 ng/mL transforming growth factor-β1 (TGF-β1). Increasing doses of Y-27632 and simvastatin were added. Real-time quantitative PCR was used to assess mRNA expression of α-smooth muscle actin (α-SMA), collagen III, elastin and connective tissue growth factor (CTGF) after 72 h. Western blot analysis was used to quantify α-SMA protein contents, and immunofluorescence (IF) was used to visualize MFB differentiation by staining for α-SMA after 72 h. A resazurin-based assay was used to assess cell viability to ensure the anti-MFB effect of the drugs. A mechanistic study was performed using IF staining for YAP/TAZ nuclear translocation. RESULTS After 72 h of stimulation with TGF-β1, a six- to 10-fold upregulation of α-SMA could be observed. When treated with Y-27632 or simvastatin, the α-SMA, collagen III, elastin and CTGF mRNA expression was impeded. Additionally, TGF-β1 stimulation showed a twofold increase in α-SMA protein expression, which was reversed to non-stimulated levels after treatment with Y-27632 and simvastatin. Using IF, stimulated cells were identified as MFB (α-SMA+, Vim+) as opposed to the non-stimulated, Y-27632- and simvastatin-treated cells (α-SMA-, Vim+). The resazurin-based assay confirmed that the cell viability was not compromised by the administered drugs. On stimulation with TGF-β1, nuclear translocation of YAP/TAZ could be observed, which was prevented by adding the aforementioned compounds. CONCLUSION Transformation of FBs into the contractile and extracellular matrix-producing MFBs occurs after TGF-β1 stimulation. In our experiments, Rho-kinase inhibition and simvastatin treatment were shown to prevent this in TGF-β1-stimulated cells on an RNA and protein level through the inhibition of YAP/TAZ nuclear translocation. Y-27632 and simvastatin could become a novel treatment option in the early treatment of PD

Colon cancer-derived myofibroblasts increase endothelial cell migration by glucocorticoid-sensitive secretion of a pro-migratory factor

AbstractAngiogenesis is important in cancer progression and can be influenced by tumor-associated myofibroblasts. We addressed the hypothesis that glucocorticoids indirectly affect angiogenesis by altering the release of pro-angiogenic factors from colon cancer-derived myofibroblasts.Our study shows that glucocorticoids reduced prostanoids, urokinase-type plasminogen activator (uPA) and angiopoietin-like protein-2 (ANGPTL2) levels, but increased angiogenin (ANG) in supernatant from human CT5.3hTERT colon cancer-derived myofibroblasts. Conditioned medium from solvent- (CMS) and dexamethasone (Dex)-treated (CMD) myofibroblasts increased human umbilical vein endothelial cell (HUVEC) proliferation, but did not affect expression of pro-angiogenic factors or tube-like structure formation (by HUVECs or human aortic ECs). In a HUVEC scratch assay CMS-induced acceleration of wound healing was blunted by CMD treatment. Moreover, CMS-induced neovessel growth in mouse aortic rings ex vivo was also blunted using CMD. The latter effect could be ascribed to both Dex-driven reduction of secreted factors and potential residual Dex present in CMD (indicated using a dexamethasone-spiked CMS control). A similar control in the scratch assay, however, revealed that altered levels of factors in the CMD, and not potential residual Dex, were responsible for decreased wound closure.In conclusion, our results suggest that glucocorticoids indirectly alter endothelial cell function during tumor development in vivo

The triterpene echinocystic acid and its 3-O-glucoside derivative are revealed as potent and selective glucocorticoid receptor agonists

Glucocorticoids are steroid hormones widely used to control many inflammatory conditions. These effects are primarily attributed to glucocorticoid receptor transrepressional activities but with concomitant receptor transactivation associated with considerable side effects. Accordingly, there is an immediate need for selective glucocorticoid receptor agonists able to dissociate transactivation from transrepression. Triterpenoids have structural similarities with glucocorticoids and exhibit anti-inflammatory and apoptotic activities via mechanisms that are not well-defined. In this study, we examined whether echinocystic acid and its 3-O-glucoside derivative act, at least in part, through the regulation of glucocorticoid receptor and whether they can constitute selective receptor activators. We showed that echinocystic acid and its glucoside induced glucocorticoid receptor nuclear translocation by 75% and 55%. They suppressed the nuclear factor-kappa beta transcriptional activity by 20% and 70%, respectively, whereas they have no glucocorticoid receptor transactivation capability and stimulatory effect on the expression of the phosphoenolopyruvate carboxykinase target gene in HeLa cells. Interestingly, their suppressive effect is diminished in glucocorticoid receptor low level COS-7 cells, verifying the receptor involvement in this process. Induced fit docking calculations predicted favorable binding in the ligand binding domain and structural characteristics which can be considered consistent with the experimental observations. Further, glucocorticoids exert apoptotic activities; we have demonstrated here that the echinocystic acids in combination with the synthetic glucocorticoid, dexamethasone, induce apoptosis. Taken together, our results indicate that echinocystic acids are potent glucocorticoid receptor regulators with selective transrepressional activities (dissociated from transactivation), highlighting the potential of echinocystic acid derivatives as more promising treatments for inflammatory conditions