Up-Regulation of RACK1 by TGF-β1 Promotes Hepatic Fibrosis in Mice

Liver fibrosis represents the consequences of a sustained wound healing response to chronic liver injury, and activation of quiescent hepatic stellate cells (HSCs) into a myofibroblast-like phenotype is considered as the central event of liver fibrosis. RACK1, the receptor for activated C-kinase 1, is a classical scaffold protein implicated in numerous signaling pathways and cellular processes; however, the role of RACK1 in liver fibrosis is little defined. Herein, we report that RACK1 is up-regulated in activated HSCs in transforming growth factor beta 1 (TGF-β1)-dependent manner both in vitro and in vivo, and TGF-β1 stimulates the expression of RACK1 through NF-κB signaling. Moreover, RACK1 promotes TGF-β1 and platelet-derived growth factor (PDGF)-mediated activation of pro-fibrogenic pathways as well as the differentiation, proliferation and migration of HSCs. Depletion of RACK1 suppresses the progression of TAA-induced liver fibrosis in vivo. In addition, the expression of RACK1 in fibrogenic cells also positively correlates well with the stage of liver fibrosis in clinical cases. Our results suggest RACK1 as a downstream target gene of TGF-β1 involved in the modulation of liver fibrosis progression in vitro and in vivo, and propose a strategy to target RACK1 for liver fibrosis treatment.     

Loss of β1-Integrin Enhances TGF-β1-induced Collagen Expression in Epithelial Cells via Increased αvβ3-Integrin and Rac1 Activity

Transforming growth factor β (TGF-β) promotes tissue fibrosis via the receptor-specific Smad pathway and non-canonical pathways. We recently reported that TGF-β1-stimulated collagen expression by cultured kidney cells requires integrin-dependent activation of focal adhesion kinase (FAK) and consequent ERK MAP kinase activity leading to Smad3 linker region phosphorylation. Here, we defined a role for αvβ3-integrin in this non-canonical pathway. A human kidney tubular cell line in which β1-integrin was knocked down (β1-k/d) demonstrated enhanced type I collagen mRNA expression and promoter activity. A second shRNA to either αv-integrin or β3-integrin, but not to another αv-binding partner, β6-integrin, abrogated the enhanced COL1A2 promoter activity in β1-k/d cells. Although αvβ3-integrin surface expression levels were not different, αvβ3-integrins colocalized with sites of focal adhesion significantly more in β1-k/d cells, and activated αvβ3-integrin was detected only in β1-k/d cells. Further, the collagen response was decreased by a function-blocking antibody or a peptide inhibitor of αvβ3-integrin. In cells lacking αvβ3-integrin, the responses were attenuated, whereas the response was enhanced in αvβ3-overexpressing cells. Rac1 and ERK, previously defined mediators for this non-canonical pathway, showed increased activities in β1-k/d cells. Finally, inhibition of αvβ3-integrin decreased Rac1 activity and COL1A2 promoter activity in β1-k/d cells. Together, our results indicate that decreasing β1 chain causes αvβ3-integrin to become functionally dominant and promotes renal cell fibrogenesis via Rac1-mediated ERK activity.     

RNF-121 Is an Endoplasmic Reticulum-Membrane E3 Ubiquitin Ligase Involved in the Regulation of β-Integrin

We report on the characterization of RNF-121, an evolutionarily conserved E3 ligase RING finger protein that is expressed in the endoplasmic reticulum (ER) of various cells and tissues in Caenorhabditis elegans. Inactivation of RNF-121 induced an elevation in BiP expression and increased the sensitivity of worms to ER stress. Genetic analysis placed RNF-121 downstream of the unfolded protein response (UPR) regulator protein kinase-like endoplasmic reticulum kinase (PERK). We identify PAT-3::GFP, the β subunit of the heterodimeric integrin receptors, as an RNF-121 substrate; whereas induction of RNF-121 expression reduced the level of PAT-3::GFP in the gonad distal tip cells, inhibition of RNF-121 led to the accumulation of stably bound PAT-3::GFP inclusions. Correspondingly, overexpression of RNF-121 during early stages of gonad development led to aberrations in germline development and gonad migration that overlap with those observed after PAT-3 inactivation. The formation of these gonad abnormalities required functional ER-associated degradation (ERAD) machinery. Our findings identify RNF-121 as an ER-anchored ubiquitin ligase that plays a specific role in the ERAD pathway by linking it to the regulation of the cell adhesion integrin receptors.     

Attenuation of CCl4-Induced Hepatic Fibrosis in Mice by Vaccinating against TGF-β1

Transforming growth factor β1 (TGF-β1) is the pivotal pro-fibrogenic cytokine in hepatic fibrosis. Reducing the over-produced expression of TGF-β1 or blocking its signaling pathways is considered to be a promising therapeutic strategy for hepatic fibrosis. In this study, we evaluated the feasibility of attenuating hepatic fibrosis by vaccination against TGF-β1 with TGF-β1 kinoids. Two TGF-β1 kinoid vaccines were prepared by cross-linking TGF-β1-derived polypeptides (TGF-β1²⁵–[41-65] and TGF-β1³⁰–[83-112]) to keyhole limpet hemocyanin (KLH). Immunization with the two TGF-β1 kinoids efficiently elicited the production of high-levels of TGF-β1-specific antibodies against in BALB/c mice as tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The antisera neutralized TGF-β1-induced growth-inhibition on mink lung epithelial cells (Mv1Lu) and attenuated TGF-β1-induced Smad2/3 phosphorylation, α-SMA, collagen type 1 alpha 2 (COL1A2), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in the rat hepatic stellate cell (HSC) line, HSC-T6. Vaccination against TGF-β1 with the kinoids significantly suppressed CCl₄-induced collagen deposition and the expression of α-SMA and desmin, attenuated hepatocyte apoptosis and accelerated hepatocyte proliferation in BALB/c mice. These results demonstrated that immunization with the TGF-β1 kinoids efficiently attenuated CCl₄-induced hepatic fibrosis and liver injury. Our study suggests that vaccination against TGF-β1 might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases.     

Endothelial-Rac1 Is Not Required for Tumor Angiogenesis unless αvβ3-Integrin Is Absent

Endothelial cell migration is an essential aspect of tumor angiogenesis. Rac1 activity is needed for cell migration in vitro implying a requirement for this molecule in angiogenesis in vivo. However, a precise role for Rac1 in tumor angiogenesis has never been addressed. Here we show that depletion of endothelial Rac1 expression in adult mice, unexpectedly, has no effect on tumor growth or tumor angiogenesis. In addition, repression of Rac1 expression does not inhibit VEGF-mediated angiogenesis in vivo or ex vivo, nor does it affect chemotactic migratory responses to VEGF in 3-dimensions. In contrast, the requirement for Rac1 in tumor growth and angiogenesis becomes important when endothelial β3-integrin levels are reduced or absent: the enhanced tumor growth, tumor angiogenesis and VEGF-mediated responses in β3-null mice are all Rac1-dependent. These data indicate that in the presence of αvβ3-integrin Rac1 is not required for tumor angiogenesis.     

Cerebral β-Amyloidosis in Mice Investigated by Ultramicroscopy

Alzheimer´s disease (AD) is the most common neurodegenerative disorder. AD neuropathology is characterized by intracellular neurofibrillary tangles and extracellular β-amyloid deposits in the brain. To elucidate the complexity of AD pathogenesis a variety of transgenic mouse models have been generated. An ideal imaging system for monitoring β-amyloid plaque deposition in the brain of these animals should allow 3D-reconstructions of β-amyloid plaques via a single scan of an uncropped brain. Ultramicroscopy makes this possible by replacing mechanical slicing in standard histology by optical sectioning. It allows a time efficient analysis of the amyloid plaque distribution in the entire mouse brain with 3D cellular resolution. We herein labeled β-amyloid deposits in a transgenic mouse model of cerebral β-amyloidosis (APPPS1 transgenic mice) with two intraperitoneal injections of the amyloid-binding fluorescent dye methoxy-X04. Upon postmortem analysis the total number of β-amyloid plaques, the β-amyloid load (volume percent) and the amyloid plaque size distributions were measured in the frontal cortex of two age groups (2.5 versus 7-8.5 month old mice). Applying ultramicroscopy we found in a proof-of-principle study that the number of β-amyloid plaques increases with age. In our experiments we further observed an increase of large plaques in the older age group of mice. We demonstrate that ultramicroscopy is a fast, and accurate analysis technique for studying β-amyloid lesions in transgenic mice allowing the 3D staging of β-amyloid plaque development. This in turn is the basis to study neural network degeneration upon cerebral β-amyloidosis and to assess Aβ -targeting therapeutics.     

TGF-β1 Regulation of Estrogen Production in Mature Rat Leydig Cells

Background

Besides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-β1 (TGF-β1) is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-β1 regulates estradiol (E₂) synthesis in adult rat Leydig cells and then to assess the impact of TGF-β1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells.

Methodology/Principal Findings

Primary cultured Leydig cells were incubated in the presence of recombinant TGF-β1 and the production of E₂ as well as testosterone (T) were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP), respectively. Results from this study show that TGF-β1 down-regulates the level of E₂ secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E₂ and TGF-β1, and E₂ treatment in turn restored the inhibition of TGF-β1 on GJIC.

Conclusions

Our results indicate, for the first time in adult rat Leydig cells, that TGF-β1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E₂ secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.     

Wound Healing Activity of Phage-Sisplayed TGF-β1 Model Peptide in Streptozotocin-Induced Diabetic Rats

International Journal of Peptide Research and Therapeutics - To evaluate the effect of phage-displayed TGF-β1 model peptide on cutaneous wound healing in streptozotocin-induced diabetic rats....     

Recruitment of β-Catenin to N-Cadherin Is Necessary for Smooth Muscle Contraction

β-Catenin is a key component that connects transmembrane cadherin with the actin cytoskeleton at the cell-cell interface. However, the role of the β-catenin/cadherin interaction in smooth muscle has not been well characterized. Here stimulation with acetylcholine promoted the recruitment of β-catenin to N-cadherin in smooth muscle cells/tissues. Knockdown of β-catenin by lentivirus-mediated shRNA attenuated smooth muscle contraction. Nevertheless, myosin light chain phosphorylation at Ser-19 and actin polymerization in response to contractile activation were not reduced by β-catenin knockdown. In addition, the expression of the β-catenin armadillo domain disrupted the recruitment of β-catenin to N-cadherin. Force development, but not myosin light chain phosphorylation and actin polymerization, was reduced by the expression of the β-catenin armadillo domain. Furthermore, actin polymerization and microtubules have been implicated in intracellular trafficking. In this study, the treatment with the inhibitor latrunculin A diminished the interaction of β-catenin with N-cadherin in smooth muscle. In contrast, the exposure of smooth muscle to the microtubule depolymerizer nocodazole did not affect the protein-protein interaction. Together, these findings suggest that smooth muscle contraction is mediated by the recruitment of β-catenin to N-cadherin, which may facilitate intercellular mechanotransduction. The association of β-catenin with N-cadherin is regulated by actin polymerization during contractile activation.     

Differential Regulation of Extracellular Matrix and Soluble Fibulin-1 Levels by TGF-β1 in Airway Smooth Muscle Cells

Fibulin-1 (FBLN-1) is a secreted glycoprotein that is associated with extracellular matrix (ECM) formation and rebuilding. Abnormal and exaggerated deposition of ECM proteins is a hallmark of many fibrotic diseases, such as chronic obstructive pulmonary disease (COPD) where small airway fibrosis occurs. The aim of this study was to investigate the regulation of FBLN-1 by transforming growth factor beta 1 (TGF-β₁) (a pro-fibrotic stimulus) in primary human airway smooth muscle (ASM) cells from volunteers with and without COPD. Human ASM cells were seeded at a density of 1×10⁴ cells/cm², and stimulated with or without TGF-β₁ (10 ng/ml) for 72 hours before FBLN-1 deposition and soluble FBLN-1 were measured. Fold change in FBLN-1 mRNA was measured at 4, 8, 24, 48, 72 hours. In some experiments, cycloheximide (0.5 µg/ml) was used to assess the regulation of FBLN-1 production. TGF-β₁ decreased the amount of soluble FBLN-1 both from COPD and non-COPD ASM cells. In contrast, the deposition of FBLN-1 into the ECM was increased in ASM cells obtained from both groups. TGF-β₁ did not increase FBLN-1 gene expression at any of the time points. There were no differences in the TGF-β₁ induced FBLN-1 levels between cells from people with or without COPD. Cycloheximide treatment, which inhibits protein synthesis, decreased both the constitutive release of soluble FBLN-1, and TGF-β₁ induced ECM FBLN-1 deposition. Furthermore, in cycloheximide treated cells addition of soluble FBLN-1 resulted in incorporation of FBLN-1 into the ECM. Therefore the increased deposition of FBLN-1 by ASM cells into the ECM following treatment with TGF-β₁ is likely due to incorporation of soluble FBLN-1 rather than de-novo synthesis.     

Regulation of the TGF-β pathway by deubiquitinases in cancer

The transforming growth factor-β (TGF-β) pathway regulates diverse cellular processes. It signals via serine/threonine kinase receptors and intracellular Smad and non-Smad effector proteins. In cancer cells, aberrant TGF-β signalling can lead to loss of growth inhibition and an increase in invasion, epithelial-to-mesenchymal transition (EMT) and metastasis. Therapeutic targeting of the pro-oncogenic TGF-β responses is currently being explored as a potential therapy against certain invasive and metastatic cancer types. The ubiquitin post-translational regulation system is emerging as a key regulatory mechanism for the control of TGF-β pathway components. In this review, we focus on the role of deubiquitinases (DUBs), which counteract the activity of E3 ubiquitin ligases. We will discuss the mechanisms by which specific DUBs control Smad and non-Smad TGF-β signalling routes, and how perturbation of the expression and function of DUBs contributes to misregulation of TGF-β signalling in cancer.     

AFAP1 Is a Novel Downstream Mediator of TGF-β1 for CCN2 Induction in Osteoblasts

Background

CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-β1 and acts as a mediator of TGF-β1 induced matrix production in osteoblasts and Src is required for CCN2 induction by TGF-β1; however, the molecular mechanisms that control CCN2 induction in osteoblasts are poorly understood. AFAP1 binds activated forms of Src and can direct the activation of Src in certain cell types, however a role for AFAP1 downstream of TGF-β1 or in osteoblats is undefined. In this study, we investigated the role of AFAP1 for CCN2 induction by TGF-β1 in primary osteoblasts.

Results

We demonstrated that AFAP1 expression in osteoblasts occurs in a biphasic pattern with maximal expression levels occurring during osteoblast proliferation (~day 3), reduced expression during matrix production/maturation (~day 14–21), an a further increase in expression during mineralization (~day 21). AFAP1 expression is induced by TGF-β1 treatment in osteoblasts during days 7, 14 and 21. In osteoblasts, AFAP1 binds to Src and is required for Src activation by TGF-β1 and CCN2 promoter activity and protein induction by TGF-β1 treatment was impaired using AFAP1 siRNA, indicating the requirement of AFAP1 for CCN2 induction by TGF-β1. We also demonstrated that TGF-β1 induction of extracellular matrix protein collagen XIIa occurs in an AFAP1 dependent fashion.

Conclusions

This study demonstrates that AFAP1 is an essential downstream signaling component of TGF-β1 for Src activation, CCN2 induction and collagen XIIa in osteoblasts.     

Regulation of IGF-1 but not TGF-β1 by NGF in the smooth muscle of the inflamed urinary bladder

Intraperitoneal injection of cyclophosphamide (CYP) causes hemorrhagic cystitis with excess growth of muscular layer leading to bladder hypertrophy; this could be attributable to changes in the expression profiles of growth factors in the inflamed urinary bladder. The growth factors characterized in the current study include nerve growth factor (NGF), insulin-like growth factor (IGF)-1, and transforming growth factor (TGF)-β1. We found that following CYP injection for 8 h and 48 h, the mRNA levels of all three factors were increased in the inflamed bladder when compared to control. The level of NGF mRNA was mainly increased in the urothelium layer while the levels of IGF-1 mRNA and TGF-β1 mRNA were increased in the smooth muscle layer. The level of NGF high affinity receptor TrkA mRNA was also increased in both the urothelium and the smooth muscle layers during bladder inflammation. When we blocked NGF action with NGF neutralizing antibody in vivo, we found that the up-regulation of IGF-1 in the inflamed bladder was reversed while the up-regulation of TGF-β1 was not affected by NGF neutralization. The effect of NGF on regulating IGF-1 expression was further confirmed in bladder smooth muscle culture showing that exogenous NGF increased the mRNA level of IGF-1 after 30 min to 1 h stimulation. These results suggested that bladder inflammation induced region-specific changes in the expression profiles of NGF, IGF-1 and TGF-β1. The up-regulation of NGF in the urothelium may have a role in affecting bladder smooth muscle cell physiology by regulating IGF-1 expression.