Extracellular Matrix Induction Research Articles

Bioactivity of human adult stem cells and functional relevance of stem cell-derived extracellular matrix in chondrogenesis

BackgroundAutologous chondrocyte implantation (ACI) has been used to treat articular cartilage defects for over two decades. Adult stem cells have been proposed as a solution to inadequate donor cell numbers often encountered in ACI. Multipotent stem/progenitor cells isolated from adipose, bone marrow, and cartilage are the most promising cell therapy candidates. However, different essential growth factors are required to induce these tissue-specific stem cells to initiate chondrogenic differentiation and subsequent deposition of extracellular matrix (ECM) to form cartilage-like tissue. Upon transplantation into cartilage defects in vivo, the levels of growth factors in the host tissue are likely to be inadequate to support chondrogenesis of these cells in situ. The contribution of stem/progenitor cells to cartilage repair and the quality of ECM produced by the implanted cells required for cartilage repair remain largely unknown. Here, we evaluated the bioactivity and chondrogenic induction ability of the ECM produced by different adult stem cells.MethodsAdult stem/progenitor cells were isolated from human adipose (hADSCs), bone marrow (hBMSCs), and articular cartilage (hCDPCs) and cultured for 14 days in monolayer in mesenchymal stromal cell (MSC)–ECM induction medium to allow matrix deposition and cell sheet formation. The cell sheets were then decellularized, and the protein composition of the decellularized ECM (dECM) was analyzed by BCA assay, SDS-PAGE, and immunoblotting for fibronectin (FN), collagen types I (COL1) and III (COL3). The chondrogenic induction ability of the dECM was examined by seeding undifferentiated hBMSCs onto the respective freeze-dried solid dECM followed by culturing in serum-free medium for 7 days. The expression levels of chondrogenic genes SOX9, COL2, AGN, and CD44 were analyzed by q-PCR.ResultshADSCs, hBMSCs, and hCDPCs generated different ECM protein profiles and exhibited significantly different chondrogenic effects. hADSCs produced 20–60% more proteins than hBMSCs and hCDPCs and showed a fibrillar-like ECM pattern (FNhigh, COL1high). hCDPCs produced more COL3 and deposited less FN and COL1 than the other cell types. The dECM derived from hBMSCs and hCDPCs induced spontaneous chondrogenic gene expression in hBMSCs.ConclusionsThese findings provide new insights on application of adult stem cells and stem cell-derived ECM to enhance cartilage regeneration.

Calreticulin exploits TGF-β for extracellular matrix induction engineering a tissue regenerative process.

Topical application of extracellular calreticulin (eCRT), an ER chaperone protein, in animal models enhances wound healing and induces tissue regeneration evidenced by epidermal appendage neogenesis and lack of scarring. In addition to chemoattraction of cells critical to the wound healing process, eCRT induces abundant neo-dermal extracellular matrix (ECM) formation by 3days post-wounding. The purpose of this study was to determine the mechanisms involved in eCRT induction of ECM. In vitro, eCRT strongly induces collagen I, fibronectin, elastin, α-smooth muscle actin in human adult dermal (HDFs) and neonatal fibroblasts (HFFs) mainly via TGF-β canonical signaling and Smad2/3 activation; RAP, an inhibitor of LRP1 blocked eCRT ECM induction. Conversely, eCRT induction of α5 and β1 integrins was not mediated by TGF-β signaling nor inhibited by RAP. Whereas eCRT strongly induces ECM and integrin α5 proteins in K41 wild-type mouse embryo fibroblasts (MEFs), CRT null MEFs were unresponsive. The data show that eCRT induces the synthesis and release of TGF-β3 first via LRP1 or other receptor signaling and later induces ECM proteins via LRP1 signaling subsequently initiating TGF-β receptor signaling for intracellular CRT (iCRT)-dependent induction of TGF-β1 and ECM proteins. In addition, TGF-β1 induces 2-3-fold higher level of ECM proteins than eCRT. Whereas eCRT and iCRT converge for ECM induction, we propose that eCRT attenuates TGF-β-mediated fibrosis/scarring to achieve tissue regeneration.

Neutrophil proteome shifts over the myocardial infarction time continuum

In response to myocardial infarction (MI), neutrophils (PMNs) are early responders that initiate the inflammatory reaction. Because macrophages and fibroblasts show polarization states after MI, we hypothesized PMNs also undergo phenotypic changes over the MI time course. The objective of the current study was to map the continuum of polarization phenotypes in cardiac neutrophils over the first week of MI. C57BL/6J male mice (3–6 months old) underwent permanent coronary artery ligation to induce MI, and PMNs were isolated from the infarct region at days 1, 3, 5, and 7 after MI. Day 0 served as a no MI negative control. Aptamer proteomics was performed on biological replicates (n = 10–12) for each time point. Day (D)1 MI neutrophils had a high degranulation profile with increased matrix metalloproteinase (MMP) activity. D3 MI neutrophil profiles showed upregulation of apoptosis and induction of extracellular matrix (ECM) organization. D5 MI neutrophils further increased their ECM reorganization profile. D7 MI neutrophils had a reparative signature that included expression of fibronectin, galectin-3, and fibrinogen to contribute to scar formation by stimulating ECM reorganization. Of note, fibronectin was a key modulator of degranulation, as it amplified MMP-9 release in the presence of an inflammatory stimulus. Our results indicate that neutrophils selectively degranulate over the MI time course, reflective of both their intrinsic protein profiles as well as the ECM environment in which they reside. MMPs, cathepsins, and ECM proteins were prominent neutrophil degranulation indicators.

Role of Calbindin-D28k in Diabetes-Associated Advanced Glycation End-Products-Induced Renal Proximal Tubule Cell Injury

Diabetes-associated advanced glycation end-products (AGEs) can increase extracellular matrix (ECM) expression and induce renal fibrosis. Calbindin-D28k, which plays a role in calcium reabsorption in renal distal convoluted tubules, is increased in a diabetic kidney. The role of calbindin-D28k in diabetic nephropathy still remains unclear. Here, calbindin-D28k protein expression was unexpectedly induced in the renal tubules of db/db diabetic mice. AGEs induced the calbindin-D28k expression in human renal proximal tubule cells (HK2), but not in mesangial cells. AGEs induced the expression of fibrotic molecules, ECM proteins, epithelial-mesenchymal transition (EMT) markers, and endoplasmic reticulum (ER) stress-related molecules in HK2 cells, which could be inhibited by a receptor for AGE (RAGE) neutralizing antibody. Calbindin-D28k knockdown by siRNA transfection reduced the cell viability and obviously enhanced the protein expressions of fibrotic factors, EMT markers, and ER stress-related molecules in AGEs-treated HK2 cells. Chemical chaperone 4-Phenylbutyric acid counteracted the AGEs-induced ER stress and ECM and EMT markers expressions. Calbindin-D28k siRNA in vivo delivery could enhance renal fibrosis in db/db diabetic mice. These findings suggest that inducible calbindin-D28k protects against AGEs/RAGE axis-induced ER stress-activated ECM induction and cell injury in renal proximal tubule cells.

PTEN improve renal fibrosis in vitro and in vivo through inhibiting FAK/AKT signaling pathway.

Renal fibrosis, the ultimate common pathway of progressive nephropathy, is characterized by excess accumulation and deposition of extracellular matrix (ECM) within the renal interstitium and glomeruli, finally resulting in end-stage kidney failure. TGFβ1 is not only abnormally increased during fibrosis but also involved in ECM induction and accumulation. Based on the bioinformative analyses, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and focal adhesion kinase (FAK) signaling pathway might be involved in TGFβ1 functions on renal fibrosis development. In the present study, fibrosis was induced in HK-2 cells using TGFβ1 and PTEN expression was significantly suppressed by 24 or 48 hours TGFβ1 treatment. PTEN overexpression in HK-2 cells improved TGFβ1-induced fibrosis within α-SMA and E-cadherin. According to the KEGG signaling pathway annotation analyses on microarray profiles (GSE23338 and GSE20247) and immunoblotting validation, FAK signaling might be involved in PTEN functions in TGFβ1-induced fibrosis. PTEN overexpression significantly inhibited TGFβ1- or unilateral ureteral obstruction (UUO)-induced FAK signaling pathway activation both in vitro and in vivo; more importantly, PTEN silence enhanced TGFβ1- or UUO-induced fibrosis, while FAK inhibitor PF567721 significantly reversed the effects of PTEN silence, indicating that PTEN exerted its effects on TGFβ1- and UUO-induced fibrotic development in vitro and in vivo via inhibiting FAK signaling pathway. In summary, these findings indicate that PTEN could improve cellular fibrotic changes and renal fibrosis via inhibiting FAK/AKT signaling pathway. Restoring PTEN expression to target FAK/AKT signaling pathway might be a potent strategy for renal fibrosis treatment.

Adapting extracellular matrix proteomics for clinical studies on cardiac remodeling post-myocardial infarction.

Following myocardial infarction (MI), the left ventricle (LV) undergoes a series of cardiac wound healing responses that involve stimulation of robust inflammation to clear necrotic myocytes and tissue debris and induction of extracellular matrix (ECM) protein synthesis to generate a scar. Proteomic strategies provide us with a means to index the ECM proteins expressed in the LV, quantify amounts, determine functions, and explore interactions. This review will focus on the efforts taken in the proteomics research field that have expanded our understanding of post-MI LV remodeling, concentrating on the strengths and limitations of different proteomic approaches to glean information that is specific to ECM turnover in the post-MI setting. We will discuss how recent advances in sample preparation and labeling protocols increase our successes at detecting components of the cardiac ECM proteome. We will summarize how proteomic approaches, focusing on the ECM compartment, have progressed over time to current gel-free methods using decellularized fractions or labeling strategies that will be useful for clinical applications. This review will provide an overview of how cardiac ECM proteomics has evolved over the last decade and will provide insight into future directions that will drive forward our understanding of cardiac ECM turnover in the post-MI LV.

MMP-9 signaling in the left ventricle following myocardial infarction.

Following myocardial infarction (MI), the left ventricle (LV) undergoes a series of cardiac wound healing responses that involve both the stimulation of robust inflammation to clear necrotic myocytes and tissue debris and the induction of extracellular matrix (ECM) protein synthesis to generate an infarct scar. The collective changes in myocardial structure and function are termed LV remodeling, and matrix metalloproteinase-9 (MMP-9) is a key instigator of post-MI LV remodeling. Through direct molecular effects on ECM and inflammatory protein turnover as well as indirect effects on major cell types that coordinate cardiac wound healing, namely the infiltrating leukocytes and the cardiac fibroblasts, MMP-9 coordinates multiple aspects of LV remodeling. In this review, we will discuss recent research that has expanded our understanding of post-MI LV remodeling, including recent proteomic advances focused on the ECM compartment to provide novel functional and translational insights. This overview will summarize how our understanding of MMP-9 has evolved over the last decade and will provide insight into future directions that will drive our understanding of MMP-9-directed cardiac ECM turnover in the post-MI LV.

Investigation of cell seeding and culture conditions for vascular smooth muscle cell culture on a degradable polyurethane scaffold

Event Abstract Back to Event Investigation of cell seeding and culture conditions for vascular smooth muscle cell culture on a degradable polyurethane scaffold Xiaoqing Zhang1, Kyle Battiston1, Craig Simmons1, 2, 3 and Paul Santerre1, 3 1 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada 2 University of Toronto, Department of Mechanical and Industrial Engineering, Canada 3 University of Toronto, Faculty of Dentistry, Canada Introduction: The development of tissue engineered vascular grafts (TEVGs) requires large quantities of uniformly distributed vascular smooth muscle cells (VSMCs) in biomaterial scaffolds, VSMC proliferation, and induction of extracellular matrix (ECM) production[1]. A biodegradable polyurethane scaffold (D-PHI) has been shown to support an anti-inflammatory phenotype for monocytes, which are one of the first cells to migrate to an implant site and provide vital signalling for wound healing and new tissue regeneration[2]. Vitamin C (Vc) derivatives ( ascorbic acid (AA) and sodium ascorbate (SA)), have been shown to stimulate VSMC growth and ECM production, and modulate cytokine-release from monocytes[3]-[6]. The objective of the current study was to investigate cell seeding strategies on D-PHI, and to use the identified conditions to compare AA and SA with respect to supporting ECM production and maintaining a desired VSMC phenotype. Materials and Methods: D-PHI scaffolds were prepared using a previously established protocol[7]. Human coronary artery SMC (Lonza CC-2583) seeding optimization was performed on D-PHI disk-formed scaffolds (6 mm diameter, 1 mm thickness) to find the maximum number of cells that could be seeded as a function of time, frequency of seeding, and the seeding volume. Seeded scaffolds were analyzed for total DNA and stained by hematoxylin and eosin (H&E) for histology. 300,000 (300k) VSMCs were seeded onto each disk scaffold and treated with 50 µM, 100µM or 200µM AA or SA for 1 week and characterized for DNA and ECM production (glycosaminoglycans (GAG), collagen and elastin). Data were analyzed by analysis of variance (SPSS 17.0). Statistical significance was reported for p < 0.05. Results and Discussion: The highest scaffold seeding efficiency was achieved with 75k or 150k VSMCs (Fig 1A). However, scaffolds seeded with 75k up to 300k VSMCs all demonstrated good cell infiltration into the material (Fig 1B). 300k VSMCs was selected to be used to study the optimal loading volume despite having lower seeding efficiency since it achieved the greatest number of loaded cells with good infiltration. Of the loading volumes tested, 8.3 µl allowed maximum cell uptake into the porous scaffolds (Fig 1C). Given that lower cell concentrations seeded with greater efficiency, a two-stage seeding protocol with 150k cells/stage, loaded over 48 hr using the optimal volume of 8.3µl, was assessed (Fig. 1). The final seeding efficiency of this latter approach was close to 85% (Fig 1D). AA and SA comparative data are reported in Fig. 2 and show that SA outperformed AA in inducing GAG (50 µM, 100µM and 200µM) and elastin (100µM and 200µM) production. Conclusion: This study has identified important seeding conditions that improved cellularity in D-PHI scaffolds. When used to compare AA vs SA on DNA and ECM protein production it was found that SA outperformed AA. These conditions will be used for future TEVG development in co-culture with human monocytes. Canadian Institutes of Health Research operating grant #230762.

Underlying molecular mechanisms of DIO2 susceptibility in symptomatic osteoarthritis

ObjectivesTo investigate how the genetic susceptibility gene DIO2 confers risk to osteoarthritis (OA) onset in humans and to explore whether counteracting the deleterious effect could contribute to novel therapeutic approaches.MethodsEpigenetically...

Calreticulin Regulates Transforming Growth Factor-β-stimulated Extracellular Matrix Production

Endoplasmic reticulum (ER) stress is an emerging factor in fibrotic disease, although precise mechanisms are not clear. Calreticulin (CRT) is an ER chaperone and regulator of Ca(2+) signaling up-regulated by ER stress and in fibrotic tissues. Previously, we showed that ER CRT regulates type I collagen transcript, trafficking, secretion, and processing into the extracellular matrix (ECM). To determine the role of CRT in ECM regulation under fibrotic conditions, we asked whether CRT modified cellular responses to the pro-fibrotic cytokine, TGF-β. These studies show that CRT-/- mouse embryonic fibroblasts (MEFs) and rat and human idiopathic pulmonary fibrosis lung fibroblasts with siRNA CRT knockdown had impaired TGF-β stimulation of type I collagen and fibronectin. In contrast, fibroblasts with increased CRT expression had enhanced responses to TGF-β. The lack of CRT does not impact canonical TGF-β signaling as TGF-β was able to stimulate Smad reporter activity in CRT-/- MEFs. CRT regulation of TGF-β-stimulated Ca(2+) signaling is important for induction of ECM. CRT-/- MEFs failed to increase intracellular Ca(2+) levels in response to TGF-β. NFAT activity is required for ECM stimulation by TGF-β. In CRT-/- MEFs, TGF-β stimulation of NFAT nuclear translocation and reporter activity is impaired. Importantly, CRT is required for TGF-β stimulation of ECM under conditions of ER stress, as tunicamycin-induced ER stress was insufficient to induce ECM production in TGF-β stimulated CRT-/- MEFs. Together, these data identify CRT-regulated Ca(2+)-dependent pathways as a critical molecular link between ER stress and TGF-β fibrotic signaling.

Angiotensin II receptor type 1 blockade decreases CTGF/CCN2‐mediated damage and fibrosis in normal and dystrophic skeletal muscles

Connective tissue growth factor (CTGF/CCN-2) is mainly involved in the induction of extracellular matrix (ECM) proteins. The levels of CTGF correlate with the degree and severity of fibrosis in many tissues, including dystrophic skeletal muscle. The CTGF overexpression in tibialis anterior skeletal muscle using an adenoviral vector reproduced many of the features observed in dystrophic muscles including muscle damage and regeneration, fibrotic response and decrease in the skeletal muscle strength. The renin–angiotensin system is involved in the genesis and progression of fibrotic diseases through its main fibrotic components angiotensin-II and its transducer receptor AT-1. The use of AT-1 receptor blockers (ARB) has been shown to decrease fibrosis. In this paper, we show the effect of AT-1 receptor blockade on CTGF-dependent biological activity in skeletal muscle cells as well as the response to CTGF overexpression in normal skeletal muscle. Our results show that in myoblasts ARB decreased CTGF-mediated increase of ECM protein levels, extracellular signal regulated kinases 1/2 (ERK-1/2) phosphorylation and stress fibres formation. In tibialis anterior muscle overexpressing CTGF using an adenovirus, ARB treatment decreased CTGF-mediated increase of ECM molecules, α-SMA and ERK-1/2 phosphorylation levels. Quite remarkable, ARB was able to prevent the loss of contractile force of tibialis anterior muscles overexpressing CTGF. Finally, we show that ARB decreased the levels of fibrotic proteins, CTGF and ERK-1/2 phosphorylation augmented in a dystrophic skeletal muscle from mdx mice. We propose that ARB is a novel pharmacological tool that can be used to decrease the fibrosis induced by CTGF in skeletal muscle associated with muscular dystrophies.

Roles of Collagen and Periostin Expression by Cranial Neural Crest Cells during Soft Palate Development

The tissue in the palatal region can be divided into the hard and the soft palates, each having a specialized function such as occlusion, speech, or swallowing. Therefore, an understanding of the mechanism of palatogenesis in relation to the function of each region is important. However, in comparison with the hard palate, there is still a lack of information about the mechanisms of soft palate development. In this study, the authors investigated the contribution of cranial neural crest (CNC) cells to development of both hard and soft palates. They also demonstrated a unique pattern of periostin expression during soft palate development, which was closely related to that of collagen type I (Col I) in palatine aponeurosis. Furthermore, organ culture analysis showed that exogenous transforming growth factor-β (TGF-β) induced the expression of both periostin and Col I. These novel patterns of expression in the extracellular matrix (ECM) induced by CNC cells suggest that these cells may help to determine the character of both the hard and soft palates through ECM induction. TGF-β signaling appears to be one of the mediators of Col I and periostin expression in the formation of functional structures during soft palate development.

Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction

BackgroundIn chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC.MethodologyHSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3′-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting.Principal FindingsThe 3′-UTR of the collagen-1 and −4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis.ConclusionsUpregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively.

Rosiglitazone Prevents Advanced Glycation End Products-Induced Renal Toxicity Likely through Suppression of Plasminogen Activator Inhibitor-1

In the development of diabetic nephropathy, advanced glycation end products (AGEs) play a causative role via induction of extracellular matrix (ECM) accumulation. Plasminogen activator inhibitor-1 (PAI-1), as a major inhibitor of plasminogen activator that plays an important role in degrading ECM, was found to significantly increase in renal fibrotic diseases. Activation of peroxisome proliferator-activated receptor (PPAR)-gamma prevented diabetic nephropathy. The present study, therefore, was to define whether or not AGE-induced renal ECM accumulation and renal dysfunction are mediated by upregulation of PAI-1 expression and whether or not PPAR-gamma agonist can attenuate these AGE effects via suppressing PAI-1 expression. Rats were given AGEs alone by iv injection at 100 mg/kg daily with or without oral supplementation of PPAR-gamma agonist rosiglitazone (RGZ) at 2 mg/kg daily for 6 weeks. Results showed that AGEs induced a renal ECM accumulation, as shown by increases in periodic acid-Schiff-positive materials, fibronectin, and type IV collagen (Col IV) contents in glomeruli, and a mild renal dysfunction, as shown by an increase in urinary proteins. AGEs also caused an increase in PAI-1 expression and a decrease in plasminogen activator bioactivity in the kidney. Treatment with RGZ significantly ameliorated AGE-induced renal ECM accumulation, proteinuria, and PAI-1 upregulation. Direct exposure of rat mesangial cells to AGEs in vitro induced increases in fibronectin and Col IV syntheses along with an increase in PAI-1 expression, effects significantly attenuated by RGZ. Preincubation of PAI-1 antibody to AGE-treated mesangial cells completely prevented AGE-induced fibronectin and Col IV production. These results suggest that upregulation of PAI-1 expression plays a critical role in AGE-induced renal ECM accumulation. Renal protection of RGZ from AGEs may be associated with the suppression of PAI-1 expression through PPAR-dependent and independent mechanisms.

The Role and Regulation of the Nuclear Factor Kappa B Signalling Pathway in Human Labour

Within the discipline of reproductive biology, our understanding of one of the most fundamental biological processes is lacking—the cellular and molecular mechanisms that govern birth. This lack of understanding limits our ability to reduce the incidence of labour complications. The incidence of labour complications including: preterm labour; cervical incompetence; and post-date pregnancies has not diminished in decades. The key to improving the management of human labour and delivery is an understanding of how the multiple processes that are requisite for a successful labour and delivery are coordinated to achieve a timely birth. Processes of human labour include the formation of: contraction associated proteins; inflammatory mediators (e.g. cytokines); uterotonic phospholipid metabolites (e.g. prostaglandins); and the induction of extracellular matrix (ECM) remodelling. Increasingly, it is becoming evident that labour onset and birth are the result of cross-talk between multiple components of an integrated network. This hypothesis is supported by recent data implicating various upstream regulatory pathways in the control of key labour-associated processes, including the activity of enzymes involved in the formation of prostaglandins and extracellular matrix remodelling, and mediators of inflammation. Clearly, the biochemical pathways involved in the formation of these mediators represent potential sites for intervention that may translate to therapeutic interventions to delay or prevent preterm labour and delivery. Available data strongly implicate the nuclear factor-κB (NF-κB) family as candidate upstream regulators of multiple labour-associated processes. Not only do these data warrant further detailed analysis of the involvement of these pathways in the process of human labour but also promise new insights into the key mechanisms that trigger birth and the identification of new therapeutic interventions that will improve the management of labour.

Screening and identification of the up-regulated genes in human mesangial cells exposed to angiotensin II.

Accumulation of extracellular matrix (ECM) in the glomerular mesangium is a common feature of many progressive renal diseases. Angiotensin II (Ang II) plays a major role in the progression of chronic kidney diseases in part by induction of ECM. However, the precise molecular signals responsible for this effect are unknown. To explore possible molecular mechanisms of ECM production related to Ang II, we screened and identified genes up-regulated by Ang II in cultured human mesangial cells (MC). Detection of up-regulated genes was determined by mRNA populations from human MC with and without Ang II stimulation (10(-6) mol/l, 24 h) by suppression subtractive hybridization. Reverse Northern blot analysis was performed to screen for differentially expressed genes. Full-length cDNAs of three novel genes were isolated by rapid amplification of cDNA ends (RACE)-polymerase chain reaction (PCR). One of these novel genes, AngRem104, was further investigated by Northern blot, Western blot and reverse transcription (RT)-PCR. The bioinformatics analysis implied that AngRem104 coded for a nuclear protein that was widely expressed in various normal human tissues. Moreover, up-regulation of AngRem104 induced by Ang II was time-dependent and was dose-dependently blocked by the Ang II type 1 receptor antagonist, Losartan. Interestingly, we also demonstrated that AngReam104 was associated with increased fibronectin expression. We conclude that AngRem104 is a novel human gene that is related to the expression of fibronectin and that is up-regulated by Ang II in human MC. These findings may lead to new insights into the mechanisms of glomerular sclerosis associated with Ang II.

Insulin-like Growth Factor-I Induces Renal Cell Hypertrophy via a Calcineurin-dependent Mechanism

Insulin-like growth factor-I (IGF-I) may play an important role in the development of renal hypertrophy. In this study we determined the effect of IGF-I on cultured mesangial cells (MCs) and examined activation of key signaling pathways. IGF-I induced hypertrophy as determined by an increase in cell size and an increase in protein to DNA ratio and increased accumulation of extracellular matrix (ECM) proteins. IGF-I also activated both Erk1/Erk2 MAPK and phosphatidylinositol 3-kinase (PI3K) in MCs. Inhibition of either MAPK or PI3K, however, had no effect on IGF-I-induced hypertrophy or ECM production. Next, we examined the effect of IGF-I on activation of the calcium-dependent phosphatase calcineurin. IGF-I treatment stimulated calcineurin activity and increased the protein levels of calcineurin and the calcineurin binding protein, calmodulin. Cyclosporin A, an inhibitor of calcineurin, blocked both IGF-I-mediated hypertrophy and up-regulation of ECM. In addition, calcineurin resulted in sustained Akt activation, indicating possible cross-talk with other signaling pathways. Finally, IGF-I treatment resulted in the calcineurindependent nuclear localization of NFATc1. Therefore, IGF-I induces hypertrophy and increases ECM accumulation in MCs. IGF-I-mediated hypertrophy is associated with activation of Erk1/Erk2 MAPK and PI3K but does not require either of these pathways. Instead, IGF-I mediates hypertrophy via a calcineurin-dependent pathway.

Melanocyte Mediated Paracrine Induction of Extracellular Matrix Degrading Proteases in Squamous Cell Carcinoma Cells

The expression of extracellular-matrix (ECM)-degrading proteases has been shown to be necessary for invasion of tumor cells into surrounding tissue. For several tumor types, overexpression of these proteases is dependent upon interactions with adjacent fibroblast cell populations. We previously demonstrated activation of matrix metalloprotease (MMP) and urokinase-type plasminogen activator (uPa) expression in a coculture model consisting of squamous cell carcinoma cells (SCC) with dermal fibroblasts. In the present study we have examined whether melanocytes, which are known to interact closely with keratinocytes of the basal epidermal layer, might influence ECM-degrading protease expression in SCC cells as well. Upon coculture of the human SCC cell line II-4 with the nontumorigenic mouse melanocyte cell line Melan-a or treatment of II-4 cells with Melan-a conditioned media, induction of expression of the MMP matrilysin and uPa was observed. In contrast, no induction was observed for stromelysin-1 or 92-kDa type IV collagenase. Matrilysin/uPa-inducing activity was found to act at the level of gene transcription for both matrilysin and uPa and was ubiquitously expressed among six different human melanocytic cell strains/lines, ranging from primary normal melanocytes to cell lines established from metastatic melanoma lesions. These data demonstrate that melanocytic cells can exert a paracrine influence in SCC cells on the expression of specific proteases involved in ECM turnover and tumor invasiveness.