An InCytes from MBC Selection: The Exocyst Protein Sec10 Is Necessary for Primary Ciliogenesis and Cystogenesis In Vitro

Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, in which they are felt to participate in flow sensing and have been linked to the pathogenesis of cystic renal disorders such as autosomal dominant polycystic kidney disease. We previously localized the exocyst, an eight-protein complex involved in membrane trafficking, to the primary cilium of Madin-Darby canine kidney cells and showed that it was involved in cystogenesis. Here, using short hairpin RNA (shRNA) to knockdown exocyst expression and stable transfection to induce exocyst overexpression, we show that the exocyst protein Sec10 regulates primary ciliogenesis. Using immunofluorescence, scanning, and transmission electron microscopy, primary cilia containing only basal bodies are seen in the Sec10 knockdown cells, and increased ciliogenesis is seen in Sec10-overexpressing cells. These phenotypes do not seem to be because of gross changes in cell polarity, as apical, basolateral, and tight junction proteins remain properly localized. Sec10 knockdown prevents normal cyst morphogenesis when the cells are grown in a collagen matrix, whereas Sec10 overexpression results in increased cystogenesis. Transfection with human Sec10 resistant to the canine shRNA rescues the phenotype, demonstrating specificity. Finally, Par3 was recently shown to regulate primary cilia biogenesis. Par3 and the exocyst colocalized by immunofluorescence and coimmunoprecipitation, consistent with a role for the exocyst in targeting and docking vesicles carrying proteins necessary for primary ciliogenesis.     

Functional linkage between the endoplasmic reticulum protein Hsp47 and procollagen expression in human vascular smooth muscle cells

Hsp47 is a heat stress protein that interacts with procollagen in the lumen of the endoplasmic reticulum, which is vital for collagen elaboration and embryonic viability. The precise actions of Hsp47 remain unclear, however. To evaluate the effects of Hsp47 on collagen production we infected human vascular smooth muscle cells (SMCs) with a retrovirus containing Hsp47 cDNA. SMCs overexpressing Hsp47 secreted type I procollagen faster than SMCs transduced with empty vector, yielding a greater accumulation of pro alpha1(I) collagen in the extracellular milieu. Interestingly, the amount of intracellular pro alpha1(I) collagen was also increased. This was associated with an unexpected increase in the rate of pro alpha1(I) collagen chain synthesis and 2.5-fold increase in pro alpha1(I) collagen mRNA expression, without a change in fibronectin expression. This amplification of procollagen expression, synthesis, and secretion by Hsp47 imparted SMCs with an enhanced capacity to elaborate a fibrillar collagen network. The effects of Hsp47 were qualitatively distinct from, and independent of, those of ascorbate and the combination of both factors yielded an even more intricate fibril network. Given the in vitro impact of altered Hsp47 expression on procollagen production, we sought evidence for interindividual variability in Hsp47 expression and identified a common, single nucleotide polymorphism in the Hsp47 gene promoter among African Americans that significantly reduced promoter activity. Together, these findings indicate a novel means by which type I collagen production is regulated by the endoplasmic reticulum constituent, Hsp47, and suggest a potential basis for inherent differences in collagen production within the population.     

A factor from damaged rat kidney stimulates collagen biosynthesis by mesangial cells

Rats were administered CCl4, a well-defined nephrotoxin, for 20 weeks to produce glomerular sclerosis. Tubular degeneration and necrosis with interstitial fibrosis was clearly evident by histological examination. Kidneys were homogenized in phosphate-buffered saline and a collagen synthesis-stimulating factor was isolated by Sephadex G-50 gel filtration. The 5 kDa component stimulated both type I and type IV procollagen synthesis by mesangial cells and type I procollagen synthesis by rat skin fibroblasts. In each cell type, 2-6-fold increases in procollagen protein production or cell proliferation was noted. The steady-state levels of mRNA encoding for procollagen alpha 1(I) and procollagen alpha 1(IV) chains in mesangial cells were determined by by hybridization to their corresponding cDNA clones. The type I procollagen mRNA was elevated 1.4-fold compared to a 1.6-fold increase in mRNA encoding for type IV procollagen. The similar properties and chemical characteristics of this fibrogenic factor with a factor from fibrotic liver suggests they are the same and that a common endogenous collagen synthesis stimulator may be present in fibrosing organs, thus providing a driving force for collagen over-production.     

Formin mDia1 contributes to migration and epithelial-mesenchymal transition of tubular epithelial cells exposed to TGF-β1

Renal tubular epithelial cells may undergo epithelial-mesenchymal transition (EMT) in response to stimuli, such as transforming growth factor (TGF)-β1, leading to myofibroblast activation and renal fibrosis. The formin mDia1 is required for nucleation and polymerization of actin and the microtubule cytoskeleton. The present study sought to explore the role of mDia1 in EMT of tubular epithelial cells. A rat model of unilateral ureteral obstruction (UUO) was established. The expression of TGF-β1, collagen I, collagen III, and mDia1 in the kidneys was examined at day 7 after surgery. The effect of mDia1 on EMT was explored in NRK-52E cells by exposing them to TGF-β1. Increased expression of TGF-β1, collagen I, collagen III, and mDia1 was found in obstructive kidneys of UUO model rats. Exposing rat tubular epithelial cells to TGF-β1 promoted collagen I and collagen III expression but had no effect on mDia1 expression. Silencing mDia1 expression impeded epithelial cell migration as well as reduced TGF-β1, collagen, and Profilin1 expression, whereas mDia1 overexpression exerted an opposite effect. Furthermore, mDia1 regulated the expression of vimentin, α-smooth muscle actin, and E-cadherin and focal adhesion-kinase (FAK)/Src activation through Profilin1. Inhibition of the mDia1 activator RhoA by fasudil reversed EMT, and FAK/Src activation induced by mDia1. In conclusion, mDia1 regulated tubular epithelial cell migration, collagen expression, and EMT in NRK-52E cells exposed to TGF-β1. Thus, suppression of mDia1 activation might be a strategy to counteract renal fibrosis.     

Chemokine (C-X-C motif) ligand 14 contributes to lipopolysaccharide-induced fibrogenesis in mouse L929 fibroblasts via modulating PPM1A

Herein, we found that serum chemokine ligand 14 (CXCL14) was significantly enhanced in patients with idiopathic pulmonary fibrosis (IPF). In our current study, mouse L929 fibroblasts were stimulated with lipopolysaccharide (LPS) (100 ng/mL). Cell proliferation, the levels of matrix metalloproteinase 2 (MMP2) and MMP9, as well as extracellular matrix (ECM) content were assessed to evaluate the fibrogenesis of L929 cells. Proliferating cell nuclear antigen and cell viability were assessed to evaluate cell proliferation. Hydroxyproline (Hyp), collagen I/III, connective tissue growth factor (CTGF), and phosphorylated Smad2/3 (p-Smad2/3) were assessed to evaluate ECM secretion and deposition. α-Smooth muscle actin (α-SMA) was used to measure the occurrence of differentiation from fibroblast toward myofibroblast. Our data suggested that knockdown of CXCL14 prevented LPS-induced fibrogenesis of L929 cells through inhibiting cell proliferation and decreasing the expression of MMP2/9, Hyp, collagen I/III, CTGF, p-Smad2/3, and α-SMA. Notably, upregulation of protein phosphatase magnesium-dependent 1A (PPM1A) was involved in this process. On the contrary, recombinant CXCL14 protein led to an opposite effect. We first suggested that overexpression of PPM1A ameliorated LPS-induced fibrogenesis. Furthermore, we substantiated that knockdown of CXCL14 exerted an antifibrotic effect in IPF in vitro probably via the upregulation of PPM1A. Besides, evidently enhanced CXCL14, yet reduced PPM1A, was found in bleomycin-induced rat pulmonary fibrosis, confirming the roles of CXCL14 and its potential association with PPM1A in IPF in vivo. In conclusion, CXCL14 could be considered as a therapeutic target for preventing fibrogenesis of mouse L929 fibroblasts.     

Galectin-3 preserves renal tubules and modulates extracellular matrix remodeling in progressive fibrosis

Renal tubular cell apoptosis is a critical detrimental event that leads to chronic kidney injury in association with renal fibrosis. The present study was designed to investigate the role of galectin-3 (Gal-3), an important regulator of multiple apoptotic pathways, in chronic kidney disease induced by unilateral ureteral obstruction (UUO). After UUO, Gal-3 expression significantly increased compared with basal levels reaching a peak increase of 95-fold by day 7. Upregulated Gal-3 is predominantly tubular at early time points after UUO but shifts to interstitial cells as the injury progresses. On day 14, there was a significant increase in TdT-mediated dUTP nick end labeling-positive cells (129%) and cytochrome c release (29%), and a decrease in BrdU-positive cells (62%) in Gal-3-deficient compared with wild-type mice. The degree of renal damage was more extensive in Gal-3-deficient mice at days 14 and 21, 35 and 21% increase in total collagen, respectively. Despite more severe fibrosis, myofibroblasts were significantly decreased by 58% on day 14 in the Gal-3-deficient compared with wild-type mice. There was also a corresponding 80% decrease in extracellular matrix synthesis in Gal-3-deficient compared with wild-type mice. Endo180 is a recently recognized receptor for intracellular collagen degradation that is expressed by interstitial cells during renal fibrogenesis. Endo180 expression was significantly decreased by greater than 50% in Gal-3-deficient compared with wild-type mice. Taken together, these results suggested that Gal-3 not only protects renal tubules from chronic injury by limiting apoptosis but that it may lead to enhanced matrix remodeling and fibrosis attenuation.     

WNT1-inducible signaling protein-1 mediates TGF-β1-induced renal fibrosis in tubular epithelial cells and unilateral ureteral obstruction mouse models via autophagy

Renal fibrosis is a common pathway for the progression of all chronic kidney diseases to end-stage kidney disease. Studies show that WNT1-inducible signaling pathway protein-1 (WISP-1) is involved in the fibrosis of various organs. The aim of the study was to explore the functional role and potential mechanism of WISP-1 in renal fibrosis. We observed that overexpression of WISP-1 in rat tubular epithelial cells (TECs) enhanced transforming growth factor-β1 (TGF-β1)-induced production of fibrotic markers, including collagen I (Col I), fibronectin (FN) and TGF-β1, while inhibition of WISP-1 suppressed such production. In vivo, the messenger RNA and protein levels of Col I, FN, and α-smooth muscle actin were significantly inhibited after anti-WISP-1 antibody treatment for 7 days in unilateral ureteral obstruction mouse models. Moreover, blockade of WISP-1 by anti-WISP-1 antibody significantly reduced autophagy-related markers, including anti-microtubule-associated protein-1 light chain 3 (LC3) and beclin 1, while increasing sequestosome 1. In addition, overexpression of WISP-1 in TECs increased autophagy as evidenced by greater numbers of GFP-LC3 puncta and increased expression of LC3 and beclin 1 in response to TGF-β1. In contrast, knockdown of WISP-1 by small interfering RNA decreased the number of GFP-LC3 puncta and the expression of LC3 and beclin 1 in TGF-β1-treated TECs. Collectively, these data suggest that WISP-1, as a profibrotic protein, may mediate renal fibrosis by inducing autophagy in both obstructive nephropathy and TGF-β1-treated TECs. WISP-1 may serve as an effective therapeutic target for the treatment of renal fibrosis.     

Regulation of peroxisome proliferator-activated receptor-gamma in liver fibrosis

The peroxisome proliferator-activated receptors (PPARs) impart diverse cellular effects in biological systems. Because stellate cell activation during liver injury is associated with declining PPARgamma expression, we hypothesized that its expression is critical in stellate cell-mediated fibrogenesis. We therefore modulated its expression during liver injury in vivo. PPARgamma was depleted in rat livers by using an adenovirus-Cre recombinase system. PPARgamma was overexpressed by using an additional adenoviral vector (AdPPARgamma). Bile duct ligation was utilized to induce stellate cell activation and liver fibrosis in vivo; phenotypic effects (collagen I, smooth muscle alpha-actin, hydroxyproline content, etc.) were measured. PPARgamma mRNA levels decreased fivefold and PPARgamma protein was undetectable in stellate cells after culture-induced activation. During activation in vivo, collagen accumulation, assessed histomorphometrically and by hydroxyproline content, was significantly increased after PPARgamma depletion compared with controls (1.28 +/- 0.14 vs. 1.89 +/- 0.21 mg/g liver tissue, P < 0.03). In isolated stellate cells, AdPPARgamma overexpression resulted in significantly increased adiponectin mRNA expression and decreased collagen I and smooth muscle alpha-actin mRNA expression compared with controls. During in vivo fibrogenesis, rat livers exposed to AdPPARgamma had significantly less fibrosis than controls. Collagen I and smooth muscle alpha-actin mRNA expression were significantly reduced in AdPPARgamma-infected rats compared with controls (P < 0.05, n = 10). PPARgamma-deficient mice exhibited enhanced fibrogenesis after liver injury, whereas PPARgamma receptor overexpression in vivo attenuated stellate cell activation and fibrosis. The data highlight a critical role for PPARgamma during in vivo fibrogenesis and emphasize the importance of the PPARgamma pathway in stellate cells during liver injury.     

Palmitoyl ascorbate: selective augmentation of procollagen mRNA expression compared with L-ascorbate in human intestinal smooth muscle cells.

The effect of 6-O-palmitoyl ascorbate on procollagen mRNA levels, collagen synthesis, and collagen secretion was investigated and compared with the effect of L-ascorbate in human intestinal smooth muscle (HISM) cells in vitro. Collagen synthesis, determined by the incorporation of 3H-proline into pepsin-resistant, salt-precipitated collagen, increased in a concentration-dependent manner in response to palmitoyl ascorbate. There was a twofold increase in collagen synthesis at 2.5 and 5 microM. By contrast, L-ascorbate was required at 4-5 times the concentration for the same response. However, at 20 microM, both palmitoyl and L-ascorbate induced similar 2.7-fold increases in collagen synthesis. Palmitoyl ascorbate induced a 1.6- and 3.5-fold increase in steady-state levels of procollagen I and III mRNA levels respectively, whereas L-ascorbate had no effect. Palmitoyl ascorbate and L-ascorbate induced similar increases in the amounts of newly synthesized procollagen secreted into the medium and in the amounts of collagen types I, III and V accumulating in the cell layer. There was no effect of either palmitoyl ascorbate or L-ascorbate on the activity of a procollagen alpha2 (I) promoter construct transiently transfected into HISM cells. Palmitoyl ascorbate augments HISM cell procollagen synthesis and mRNA levels more efficiently than L-ascorbate. This property may be due to the greater resistance of the ascorbate ester to oxidation and suggests that palmitoyl ascorbate could be an important agent for studies of collagen synthesis in vitro.     

Up-regulation of the human-specific CHRFAM7A gene protects against renal fibrosis in mice with obstructive nephropathy

Renal fibrosis is a major factor in the progression of chronic kidney diseases. Obstructive nephropathy is a common cause of renal fibrosis, which is also accompanied by inflammation. To explore the effect of human-specific CHRFAM7A expression, an inflammation-related gene, on renal fibrosis during obstructive nephropathy, we studied CHRFAM7A transgenic mice and wild type mice that underwent unilateral ureteral obstruction (UUO) injury. Transgenic overexpression of CHRFAM7A gene inhibited UUO-induced renal fibrosis, which was demonstrated by decreased fibrotic gene expression and collagen deposition. Furthermore, kidneys from transgenic mice had reduced TGF-β1 and Smad2/3 expression following UUO compared with those from wild type mice with UUO. In addition, the overexpression of CHRFAM7A decreased release of inflammatory cytokines in the kidneys of UUO-injured mice. In vitro, the overexpression of CHRFAM7A inhibited TGF-β1-induced increase in expression of fibrosis-related genes in human renal tubular epithelial cells (HK-2 cells). Additionally, up-regulated expression of CHRFAM7A in HK-2 cells decreased TGF-β1-induced epithelial-mesenchymal transition (EMT) and inhibited activation f TGF-β1/Smad2/3 signalling pathways. Collectively, our findings demonstrate that overexpression of the human-specific CHRFAM7A gene can reduce UUO-induced renal fibrosis by inhibiting TGF-β1/Smad2/3 signalling pathway to reduce inflammatory reactions and EMT of renal tubular epithelial cells.     

The role of integrin-linked kinase in liver wound healing

Liver wound healing is an integrated process in which hepatic stellate cells play a major role. We hypothesized that the cellextracellular signaling protein integrin-linked kinase (ILK) is important in transducing signals from the extracellular matrix to stellate cells and thus plays a critical role in stellate cell activation and fibrogenesis during liver injury. Liver injury and subsequent stellate cell activation led to a 3-fold increase in ILK expression and increased kinase activity. Overexpression of ILK in isolated stellate cells led to enhanced motility and adhesion as well as increases in smooth muscle alpha-actin and type I collagen mRNA expression. The effects of ILK on stellate cell phenotypes were phosphatidylinositol 3-kinase-dependent. Forced expression of ILK in vivo led to increases in type I collagen, smooth muscle alpha-actin, transforming growth factor-beta, and extra domain A (EDA) fibronectin mRNAs (by 3.2-, 3.5-, 2.5-, and 2.2-fold, respectively; n = 8, p < 0.05 for each versus the control), whereas inhibition of ILK in vivo led to significant reductions in these mRNAs. Morphometric analysis revealed that ILK overexpression led to a 31.4% increase in liver collagen content (n = 8, p < 0.05 versus the control); in contrast ILK knockdown in vivo led to a significant reduction in fibrogenesis. We conclude that ILK plays an important pathophysiological role in vivo in liver wound healing.     

E3 ubiquitin ligase synoviolin is involved in liver fibrogenesis

Background and Aim

Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis.

Methods

The expression and localization of synoviolin in the liver were analyzed in CCl₄-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno^+/− mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno^+/− mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno^−/− mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno^−/− MEF cells.

Results

In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno^+/− mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno^+/− mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno^−/− MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.

Conclusion

Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.