The cause of fibrotic diseases, pathologies characterized by excessive production, deposition, and contraction of extracellular matrix, is unknown. To understand the molecular basis of fibrotic disease, it is essential to appreciate how matrix deposition is normally controlled and how this process is dysregulated in fibrogenesis. This review discusses the current state of knowledge concerning interactions among the profibrotic proteins transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF, CCN2), and ED-A fibronectin (ED-A FN) and the antifibrotic proteins tumor necrosis factor-α (TNF-α) and γ-interferon (IFN-γ).—Leask, A., Abraham, D. J. TGF-β signaling and the fibrotic response.

https://faseb.onlinelibrary.wiley.com/doi/pdf/10.1096/fj.03-1273rev

TGF-beta signaling and the fibrotic response.

The proteoglycan superfamily now contains more than 30 full-time molecules that fulfill a variety of biological functions. Proteoglycans act as tissue organizers, influence cell growth and the maturation of specialized tissues, play a role as biological filters and modulate growth-factor activities, regulate collagen fibrillogenesis and skin tensile strength, affect tumor cell growth and invasion, and influence corneal transparency and neurite outgrowth. Additional roles, derived from studies of mutant animals, indicate that certain proteoglycans are essential to life whereas others might be redundant. The review focuses on the most recent genetic and molecular biological studies of the matrix proteoglycans, broadly defined as proteoglycans secreted into the pericellular matrix. Special emphasis is placed on the molecular organization of the protein core, the utilization of protein modules, the gene structure and transcriptional control, and the functional roles of the various proteoglycans. When possible, proteoglycans have been grouped into distinct gene families and subfamilies offering a simplified nomenclature based on their protein core design. The structure-function relationship of some paradigmatic proteoglycans is discussed in depth and novel aspects of their biology are examined.

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Matrix Proteoglycans: From Molecular Design to Cellular Function

Although pathologic classifications exist for several renal diseases, including IgA nephropathy, focal segmental glomerulosclerosis, and lupus nephritis, a uniform classification for diabetic nephropathy is lacking. Our aim, commissioned by the Research Committee of the Renal Pathology Society, was to develop a consensus classification combining type1 and type 2 diabetic nephropathies. Such a classification should discriminate lesions by various degrees of severity that would be easy to use internationally in clinical practice. We divide diabetic nephropathy into four hierarchical glomerular lesions with a separate evaluation for degrees of interstitial and vascular involvement. Biopsies diagnosed as diabetic nephropathy are classified as follows: Class I, glomerular basement membrane thickening: isolated glomerular basement membrane thickening and only mild, nonspecific changes by light microscopy that do not meet the criteria of classes II through IV. Class II, mesangial expansion, mild (IIa) or severe (IIb): glomeruli classified as mild or severe mesangial expansion but without nodular sclerosis (Kimmelstiel-Wilson lesions) or global glomerulosclerosis in more than 50% of glomeruli. Class III, nodular sclerosis (Kimmelstiel-Wilson lesions): at least one glomerulus with nodular increase in mesangial matrix (Kimmelstiel-Wilson) without changes described in class IV. Class IV, advanced diabetic glomerulosclerosis: more than 50% global glomerulosclerosis with other clinical or pathologic evidence that sclerosis is attributable to diabetic nephropathy. A good interobserver reproducibility for the four classes of DN was shown (intraclass correlation coefficient = 0.84) in a test of this classification.

Pathologic Classification of Diabetic Nephropathy

Epithelial-mesenchymal transition (EMT), a process whereby fully differentiated epithelial cells undergo transition to a mesenchymal phenotype giving rise to fibroblasts and myofibroblasts, is increasingly recognized as playing an important role in repair and scar formation following epithelial injury. The extent to which this process contributes to fibrosis following injury in the lung is a subject of active investigation. Recently, it was demonstrated that transforming growth factor (TGF)-β induces EMT in alveolar epithelial cells (AEC) in vitro and in vivo, and epithelial and mesenchymal markers have been colocalized to hyperplastic type II (AT2) cells in lung tissue from patients with idiopathic pulmonary fibrosis (IPF), suggesting that AEC may exhibit extreme plasticity and serve as a source of fibroblasts and/or myofibroblasts in lung fibrosis. In this review, we describe the characteristic features of EMT and its mechanistic underpinnings. We further describe the contribution of EMT to fibrosis in adult tissues following injury, focusing especially on the critical role of TGF-β and its downstream mediators in this process. Finally, we highlight recent descriptions of EMT in the lung and the potential implications of this process for the treatment of fibrotic lung disease. Treatment for fibrosis of the lung in diseases such as IPF has heretofore focused largely on amelioration of potential inciting processes such as inflammation. It is hoped that this review will stimulate further consideration of the cellular mechanisms of fibrogenesis in the lung and especially the role of the epithelium in this process, potentially leading to innovative avenues of investigation and treatment.

TGF-β-induced EMT: mechanisms and implications for fibrotic lung disease

Mice provide an experimental model of unparalleled flexibility for studying mammalian diseases. Inbred strains of mice exhibit substantial differences in their susceptibility to the renal complications of diabetes. Much remains to be established regarding the course of diabetic nephropathy (DN) in mice as well as defining those strains and/or mutants that are most susceptible to renal injury from diabetes. Through the use of the unique genetic reagents available in mice (including knockouts and transgenics), the validation of a mouse model reproducing human DN should significantly facilitate the understanding of the underlying genetic mechanisms that contribute to the development of DN. Establishment of an authentic mouse model of DN will undoubtedly facilitate testing of translational diagnostic and therapeutic interventions in mice before testing in humans.

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Mouse Models of Diabetic Nephropathy

Diabetic nephropathy is characterized by excessive deposition of extracellular matrix proteins in the mesangium and basement membrane of the glomerulus and in the renal tubulointerstitium. This review summarizes the main changes in protein composition of the glomerular mesangium and basement membrane and the evidence that, in the mesangium, these are initiated by changes in glucose metabolism and the formation of advanced glycation end products. Both processes generate reactive oxygen species (ROS). The review includes discussion of how ROS may activate intracellular signaling pathways leading to the activation of redox-sensitive transcription factors. This in turn leads to change in the expression of genes encoding extracellular matrix proteins and the protease systems responsible for their turnover.

/pdf/extracellular-matrix-metabolism-in-diabetic-nephropathy-m5ttxhmkji.pdf

Extracellular Matrix Metabolism in Diabetic Nephropathy

In chronic renal diseases, progressive loss of renal function correlates with advancing tubulo-interstitial fibrosis. TGFβ1-Smad (transforming growth factor-β1–Sma and Mad protein) signalling plays an important role in the development of renal tubulo-interstitial fibrosis. Secretion of CTGF (connective-tissue growth factor; CCN2) by PTECs (proximal-tubule epithelial cells) and EMT (epithelial–mesenchymal transdifferentiation) of PTECs to myofibroblasts in response to TGFβ are critical Smad-dependent events in the development of tubulo-interstitial fibrosis. In the present study we have investigated the distinct contributions of Smad2 and Smad3 to expression of CTGF, E-cadherin, α-SMA (α-smooth-muscle actin) and MMP-2 (matrix-metalloproteinase-2) in response to TGFβ1 treatment in an in vitro culture model of HKC-8 (transformed human PTECs). RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. Cellular E-cadherin, α-SMA as well as secreted CTGF and MMP-2 were assessed by Western immunoblotting. TGFβ1 treatment induced a fibrotic phenotype with increased expression of CTGF, MMP-2 and α-SMA, and decreased expression of E-cadherin. TGFβ1-induced increases in CTGF and decreases in E-cadherin expression were Smad3-dependent, whereas increases in MMP-2 expression were Smad2-dependent. Increases in α-SMA expression were dependent on both Smad2 and Smad3 and were abolished by combined knockdown of both Smad2 and Smad3. In conclusion, we have demonstrated distinct roles for Smad2 and Smad3 in TGFβ1-induced CTGF expression and markers of EMT in human PTECs. This can be of therapeutic value in designing targeted anti-fibrotic therapies for tubulo-interstitial fibrosis.

/pdf/the-differential-role-of-smad2-and-smad3-in-the-regulation-2nwirt8531.pdf

The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFβ1 responses in human proximal-tubule epithelial cells

We characterized a rabbit polyclonal antibody raised against human recombinant connective tissue growth factor (CTGF). The antibody recognised a higher molecular mass form (approx. 56 kDa) of CTGF in mesangial cell lysates as well as the monomeric (36-38 kDa) and lower molecular mass forms (<30 kDa) reported previously. Immunohistochemistry detected CTGF protein in glomeruli of kidneys of non-obese diabetic mice 14 days after the onset of diabetes, and this was prominent by 70 days. CTGF protein is also present in glomeruli of human patients with diabetic nephropathy. No CTGF was detected in either normal murine or human glomeruli. Transient transfection of a transformed human mesangial cell line with a CTGF-V5 epitope fusion protein markedly increased fibronectin and plasminogen activator inhibitor-1 synthesis in cultures maintained in normal glucose (4 mM) conditions; a CTGF-antisense construct reduced the elevated synthesis of these proteins in high glucose (30 mM) cultures. Culture of primary human mesangial cells for 14 days in high glucose, or in low glucose supplemented with recombinant CTGF or transforming growth factor beta1, markedly increased CTGF mRNA levels and fibronectin synthesis. However, whilst co-culture with a CTGF-antisense oligonucleotide reduced the CTGF mRNA pool by greater than 90% in high glucose, it only partially reduced fibronectin mRNA levels and synthesis. A chick anti-CTGF neutralizing antibody had a similar effect on fibronectin synthesis. Thus both CTGF and CTGF-independent pathways mediate increased fibronectin synthesis in high glucose. Nevertheless CTGF expression in diabetic kidneys is likely to be a key event in the development of glomerulosclerosis by affecting both matrix synthesis and, potentially through plasminogen activator inhibitor-1, its turnover.

/pdf/role-of-connective-tissue-growth-factor-in-the-pathogenesis-3t2hop0dg8.pdf

Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy.

. Excessive deposition of fibronectin in the glomerular mesangium in diabetic nephropathy (DN) is partly due to the induction of transforming growth factor-β (TGF-β) by high glucose. TGF-β induces its downstream mediator connective tissue growth factor (CTGF), which stimulates fibronectin matrix synthesis, a process that requires the presence of α5β1 integrin. Although TGF-β has been shown to upregulate α5β1 integrin expression in human mesangial cells (HMC), little is known about the effect of CTGF on levels of this receptor. This study tested whether CTGF modulates α5β1 expression by HMC in culture and whether changes induced by TGF-β are mediated through the induction of CTGF. FACS analysis showed that both TGF-β and CTGF significantly increased cell-surface α5β1 levels compared with basal conditions. RT-PCR indicated that the changes were at the level of transcription. Treatment of cells with TGF-β and antisense CTGF oligonucleotides significantly reduced the TGF-β–induced increases in α5β1 levels. CTGF and TGF-β also significantly increased levels of ligand-occupied cell-surface β1 integrins and cell adhesion to fibronectin, the main α5β1 substrate. Antisense CTGF significantly reduced the number of adherent cells from TGF-β–stimulated cultures. Finally, α5β1 blocking antibodies inhibited HMC fibronectin matrix deposition, confirming the importance of this receptor for this process. Taken together, these data provide evidence that CTGF controls α5β1 expression by HMC in vitro . Alterations in α5β1 levels induced by TGF-β are mediated at least in part through the induction of CTGF, and specific targeting of either α5β1 or CTGF could be useful in controlling excessive fibronectin matrix production in DN. E-mail roger.mason@ic.ac.uk

CTGF Mediates TGF-β–Induced Fibronectin Matrix Deposition by Upregulating Active α5β1 Integrin in Human Mesangial Cells

Connective tissue growth factor (CTGF) is implicated as a factor promoting tissue fibrosis in several disorders, including diabetic nephropathy. However, the molecular mechanism(s) by which it functions is not known. CTGF rapidly activates several intracellular signaling molecules in human mesangial cells (HMC), including extracellular signal-related kinase 1/2, Jun NH(2)-terminal kinase, protein kinase B, CaMK II, protein kinase Calpha, and protein kinase Cdelta, suggesting that it functions via a signaling receptor. Treating HMC with CTGF stimulated tyrosine phosphorylation of proteins 75 to 80 and 140 to 180 kD within 10 min, and Western blot analysis of anti-phosphotyrosine immunoprecipitates identified the neurotrophin receptor TrkA (molecular weight approximately 140 kD). Cross-linking rCTGF to cell surface proteins with 3,3'-dithiobis(sulfosuccinimidylpropionate) revealed that complexes formed with TrkA and with the general neurotrophin co-receptor p75(NTR). rCTGF stimulated phosphorylation of TrkA (tyr 490, 674/675). K252a, a known selective inhibitor of Trk, blocked this phosphorylation, CTGF-induced activation of signaling proteins, and CTGF-dependent induction of the transcription factor TGF-beta-inducible early gene in HMC. It is concluded that TrkA serves as a tyrosine kinase receptor for CTGF.

Nadia Abdel Wahab

Papers

Extracellular Matrix Metabolism in Diabetic Nephropathy

The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFβ1 responses in human proximal-tubule epithelial cells

Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy.

CTGF Mediates TGF-β–Induced Fibronectin Matrix Deposition by Upregulating Active α5β1 Integrin in Human Mesangial Cells

Connective Tissue Growth Factor CCN2 Interacts with and Activates the Tyrosine Kinase Receptor TrkA