Regulation of Tenascin-C, a Vascular Smooth Muscle Cell Survival Factor that Interacts with the αvβ3 Integrin to Promote Epidermal Growth Factor Receptor Phosphorylation and Growth

Tenascin-C (TN-C) is induced in pulmonary vascular disease, where it colocalizes with proliferating smooth muscle cells (SMCs) and epidermal growth factor (EGF). Furthermore, cultured SMCs require TN-C for EGF-dependent growth on type I collagen. In this study, we explore the regulation and function of TN-C in SMCs. We show that a matix metalloproteinase (MMP) inhibitor (GM6001) suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-C expression in a β3 integrin– dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of EGF. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001, restores the EGF growth response and “rescues” cells from apoptosis. The mechanism by which TN-C facilitates EGF-dependent survival and growth was then investigated. We show that TN-C interactions with α_vβ₃ integrins modify SMC shape, and EGF- dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of EGF-Rs, tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF. Cross-linking SMC β₃ integrins replicates the effect of TN-C on EGF-R clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating β₃ integrin ligands in type I collagen. In turn, α_vβ₃ interactions with TN-C alter SMC shape and increase EGF-R clustering and EGF-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces apoptosis.     

Progression of human bone marrow stromal cells into both osteogenic and adipogenic lineages is differentially regulated by structural conformation of collagen I matrix via distinct signaling pathways

Adult human bone marrow stromal cells (BMSCs) containing or consisting of mesenchymal stem cells (MSCs) are an important source in tissue homeostasis and repair. Although many processes involved in their differentiation into diverse lineages have been deciphered, substantial inroads remain to be gained to synthesize a complete regulatory picture. The present study suggests that structural conformation of extracellular collagen I, the major organic matrix component in musculoskeletal tissues, plays, along with differentiation stimuli, a decisive role in the selection of differentiation lineage. It introduces a novel concept which proposes that structural transition of collagen I matrix regulates cell differentiation through distinct signaling pathways specific for the structural state of the matrix. Thus, on native collagen I matrix inefficient adipogenesis is p38-independent, whereas on its denatured counterpart, an efficient adipogenesis is primarily regulated by p38 kinase. Inversely, osteogenic differentiation occurs efficiently on native, but not on denatured collagen I matrix, with a low commencement threshold on the former and a substantially higher one on the latter. Osteogenesis on collagen I matrices in both structural conformations is fully dependent on ERK. However, whereas on native collagen I matrix osteogenic differentiation is Hsp90-dependent, on denatured collagen I matrix it is Hsp90-independent. The matrix conformation-mediated regulation appears to be one of the mechanisms determining differentiation lineage of BMSCs. It allows a novel interpretation of the bone remodeling cycle, explains the marked physiological aging-related adipogenic shift in musculoskeletal tissues, and can be a principal contributor to adipogenic shift seen in a number of clinical disorders.     

Phenotypic Switching Induced by Damaged Matrix Is Associated with DNA Methyltransferase 3A (DNMT3A) Activity and Nuclear Localization in Smooth Muscle Cells (SMC)

Extracellular matrix changes are often crucial inciting events for fibroproliferative disease. Epigenetic changes, specifically DNA methylation, are critical factors underlying differentiated phenotypes. We examined the dependency of matrix-induced fibroproliferation and SMC phenotype on DNA methyltransferases. The cooperativity of matrix with growth factors, cell density and hypoxia was also examined. Primary rat visceral SMC of early passage (0–2) were plated on native collagen or damaged/heat-denatured collagen. Hypoxia was induced with 3% O₂ (balanced 5% CO₂ and 95% N₂) over 48 hours. Inhibitors were applied 2–3 hours after cells were plated on matrix, or immediately before hypoxia. Cells were fixed and stained for DNMT3A and smooth muscle actin (SMA) or smooth muscle myosin heavy chain. Illumina 450 K array of CpG sites was performed on bisulfite-converted DNA from smooth muscle cells on damaged matrix vs native collagen. Matrix exquisitely regulates DNMT3A localization and expression, and influences differentiation in SMCs exposed to denatured matrix +/− hypoxia. Analysis of DNA methylation signatures showed that Matrix caused significant DNA methylation alterations in a discrete number of CpG sites proximal to genes related to SMC differentiation. Matrix has a profound effect on the regulation of SMC phenotype, which is associated with altered expression, localization of DNMTs and discrete changes DNA methylation.     

Adult human bone marrow stromal cells regulate expression of their MMPs and TIMPs in differentiation type-specific manner

Previously, we described a profound impact of structural conformation of collagen matrix on osteogenic and adipogenic differentiation of bone marrow stromal cells. Thus, a marginal p38-independent adipogenesis on native collagen I matrix contrasts with an efficient p38-dependent differentiation on denatured collagen I. An efficient Hsp90-dependent osteogenesis occurs on native collagen I matrix but not on its denatured counterpart where it is insignificant and proceeds in an Hsp90-independent manner. Whereas only marginal osteogenesis and no detectable adipogenesis of bone marrow stromal cells occur on native collagen IV, the same matrix supports a highly efficient adipogenesis in denatured structural state. The present study addresses the opposite direction in the flow of cell–matrix interaction, namely the cells' influence on structural state of collagen matrix, and tests the possibility that differentiating bone marrow stromal cells may adjust the expression phenotype of MMP and TIMP in such a way that, if translated into matrix modification, would facilitate the maintenance of collagen matrix in or its modification into structural state optimal for the ongoing differentiation process. The results obtained indicate that this is indeed the case. In bone marrow stromal cells stimulated to undergo adipogenesis the expression of MMP increases and that of TIMP decreases. In cells induced to undergo osteogenesis the opposite is true: MMP/TIMP expression is adjusted in a manner that, if translated into matrix modification, could promote the native structural conformation optimal for this type of differentiation. The results obtained also indicate that the observed adjustment in MMP/TIMP expression phenotype might be an early differentiation event and that differentiation stimulation alone might be sufficient to trigger it even on matrices not favorable to a given type of differentiation. The findings of the present study raise significant questions and indicate directions for further experimentation.     

Soluble Integrin Ligands and Growth Factors Independently Rescue Neuroblastoma Cells from Apoptosis under Nonadherent Conditions

We have investigated the role of extracellular matrix (ECM) and growth factors in the survival of nonadherent human neuroblastoma cells (line SK-N-BE). Cells cultured in serum-free medium under nonadherent conditions died with apoptotic-like features (cromatin condensation and nuclear fragmentation). SK-N-BE cells underwent neuronal differentiation in response to retinoic acid (RA). While RA itself did not induce apoptosis, differentiation increased the susceptibility of SK-N-BE cells to detachment-induced apoptosis. The appearance of the apoptotic-like phenotype required the maintenance in suspension of SK-N-BE cells for at least 16 h (12.43 ± 1.40% of cells undergoing apoptosis) and the percentage increased up to 46.84 ± 3.15% after 24 h. Suspension-induced apoptosis did not depend on increased intracellular Ca²⁺levels nor onde novoprotein synthesis and was not associated with extensive DNA degradation. Stimulation by soluble collagen I rescued suspended cells from apoptosis, even in the absence of cell adhesion and spreading. The survival promoting effect of ECM was mediated by the integrin receptors, since ([1]) the protective effect of soluble collagen I was blocked by anti-integrin antibodies to β₁and α₁subunits and ([2]) the antibody-induced clustering of α₁, α₃, α_v, β₁, and β₃integrins rescued SK-N-BE cells cultured in suspension from apoptosis. As expected, adhesion on immobilized ECM proteins, collagen I, or laminin (0.1 to 10 μg/ml) also rescued SK-N-BE cells from apoptosis in a dose-dependent manner. Thede novoprotein synthesis was required to promote the survival effect of ECM, since cycloheximide completely abolished the protective effect of collagen I and protection from apoptosis by ECM or by anti-β₁antibody was associated with the increased expression ofbcl-2.In addition to integrin stimulation, serum, insulin, and nerve growth factor inhibited suspension-induced apoptosis of SK-N-BE cells. The survival effect of serum and growth factors did not require the synthesis of new proteins, unlike the ECM effect. These data show that matrix proteins can promote cell survival in neuronal cells via integrin receptors. This effect does not require cell adhesion and the subsequent changes in cell shape as it can be mediated by soluble integrin ligands in suspended cells and involves a signaling pathway different from that triggered by growth factors.     

Hsp90 chaperones PPARγ and regulates differentiation and survival of 3T3-L1 adipocytes

Adipose tissue dysregulation has a major role in various human diseases. The peroxisome proliferator-activated receptor-γ (PPARγ) is a key regulator of adipocyte differentiation and function, as well as a target of insulin-sensitizing drugs. The Hsp90 chaperone stabilizes a diverse set of signaling ‘client'' proteins, thereby regulates various biological processes. Here we report a novel role for Hsp90 in controlling PPARγ stability and cellular differentiation. Specifically, we show that the Hsp90 inhibitors geldanamycin and novobiocin efficiently impede the differentiation of murine 3T3-L1 preadipocytes. Geldanamycin at higher concentrations also inhibits the survival of both developing and mature adipocytes, respectively. Further, Hsp90 inhibition disrupts an Hsp90-PPARγ complex, leads to the destabilization and proteasomal degradation of PPARγ, and inhibits the expression of PPARγ target genes, identifying PPARγ as an Hsp90 client. A similar destabilization of PPARγ and a halt of adipogenesis also occur in response to protein denaturing stresses caused by a single transient heat-shock or proteasome inhibition. Recovery from stress restores PPARγ stability and adipocyte differentiation. Thus, our findings reveal Hsp90 as a critical stress-responsive regulator of adipocyte biology and offer a potential therapeutic target in obesity and the metabolic syndrome.     

Interactions between Cells and Collagen V Molecules or Single Chains Involve Distinct Mechanisms

Acid-soluble and pepsin-treated collagen V were prepared from fetal human bones or human placenta, respectively, to be tested for potential cell adhesion promoting activity. Out of 14 different collagen I-adhering cell lines, 10 showed distinct adhesion to collagen V. In all cases adhesion was followed by spreading. The activities of intact and pepsin-solubilized collagen V were similar, suggesting that the cell binding sites are restricted to the triple-helical domain of the molecules. Cell adhesion was also induced by the unfolded form of collagen V and after separation of the α chains by heparin affinity chromatography. Isolated α2(V) chains, rich in RGD sequences, were more efficient than isolated α1(V) chains. However, cell adhesion to native or denatured collagen V did not proceed by the same molecular mechanisms as shown by cell adhesion inhibition experiments. Cell adhesion to native collagen V was insensitive to the presence of RGD-containing synthetic peptides while adhesion to denatured collagen V was inhibited by the peptides. Furthermore, the results strongly suggested a major role for α1α1 and α2β1 integrins in the RGD-independent cell adhesion to native collagen V. These data indicate that collagen V is a specific adhesive substrate for different cell types. It also suggests that distinct sets of RGD-dependent and RGD-independent receptors mediate cell attachment to unfolded and native collagen V, respectively. This mechanism is shared by at least the interstitial collagens I and VI, which supports the hypothesis that when included in the triple-helical conformation of collagens, RGD sequences are either not accessible to cells or exhibit specific conformations recognized by different integrins.     

The Activity of Collagenase-1 Is Required for Keratinocyte Migration on a Type I Collagen Matrix

We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti–MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the α2 integrin subunit but not by antibodies against the α1 or α3 subunits. We propose that interaction of the α2β1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.     

An abundant chick gizzard integrin is the avian α1β1 integrin heterodimer and functions as a divalent cation-dependent collagen IV receptor

The α-subunit of an abundant chick gizzard integrin was isolated ([12.], J. Biol. Chem. 262, 17,189–17,199) and fragmented by proteolytic digestion. The N-terminal sequences of the intact polypeptide and of several internal peptides were determined and were found to be highly homologous to the mammalian integrin α₁-subunit. Monoclonal antibodies to the chick integrin β₁-chain react on immunoblots with the gizzard integrin β-subunit ([28.], J. Biol. Chem. 265, 14,561–14,565). The chain composition of the abundant chick gizzard integrin is therefore α₁β₁. Polyclonal antibodies to the avian integrin α₁-subunit block attachment of embryonic gizzard cells to human and chick collagen IV completely and inhibit attachment to mouse Engelbreth-Holm-Swarm (EHS) tumor laminin partially. In ELISA-style receptor assays, the isolated α₁β₁ integrin bound to human and chick collagen IV and to mouse EHS tumor and chick heart laminin. While the binding to collagen IV was abolished by removal of divalent cations, the binding to laminin was not sensitive to EDTA under the conditions used. Collagen I bound the isolated avian α₁β₁ integrin only weakly. As collagen IV was the only extracellular matrix protein for which a consistent, divalent cation-dependent, binding to the avian α₁β₁ integrin could be demonstrated in both cellular and molecular assays we suggest that it is a preferred ligand for this integrin.     

Arsenic trioxide inhibits transforming growth factor-β1-induced fibroblast to myofibroblast differentiation in vitro and bleomycin induced lung fibrosis in vivo

Background

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of insidious onset, and is responsible for up to 30,000 deaths per year in the U.S. Excessive production of extracellular matrix by myofibroblasts has been shown to be an important pathological feature in IPF. TGF-β1 is expressed in fibrotic lung and promotes fibroblast to myofibroblast differentiation (FMD) as well as matrix deposition.

Methods

To identify the mechanism of Arsenic trioxide’s (ATO)’s anti-fibrotic effect in vitro, normal human lung fibroblasts (NHLFs) were treated with ATO for 24 hours and were then exposed to TGF-β1 (1 ng/ml) before harvesting at multiple time points. To investigate whether ATO is able to alleviate lung fibrosis in vivo, C57BL/6 mice were administered bleomycin by oropharyngeal aspiration and ATO was injected intraperitoneally daily for 14 days. Quantitative real-time PCR, western blotting, and immunofluorescent staining were used to assess the expression of fibrotic markers such as α-smooth muscle actin (α-SMA) and α-1 type I collagen.

Results

Treatment of NHLFs with ATO at very low concentrations (10-20nM) inhibits TGF-β1-induced α-smooth muscle actin (α-SMA) and α-1 type I collagen mRNA and protein expression. ATO also diminishes the TGF-β1-mediated contractile response in NHLFs. ATO’s down-regulation of profibrotic molecules is associated with inhibition of Akt, as well as Smad2/Smad3 phosphorylation. TGF-β1-induced H₂O₂ and NOX-4 mRNA expression are also blocked by ATO. ATO-mediated reduction in Smad3 phosphorylation correlated with a reduction of promyelocytic leukemia (PML) nuclear bodies and PML protein expression. PML-/- mouse embryonic fibroblasts (MEFs) showed decreased fibronectin and PAI-1 expression in response to TGF-β1. Daily intraperitoneal injection of ATO (1 mg/kg) in C57BL/6 mice inhibits bleomycin induced lung α-1 type I collagen mRNA and protein expression.

Conclusions

In summary, these data indicate that low concentrations of ATO inhibit TGF-β1-induced fibroblast to myofibroblast differentiation and decreases bleomycin induced pulmonary fibrosis.     

Human bone marrow-derived stromal cells show highly efficient stress-resistant adipogenesis on denatured collagen IV matrix but not on its native counterpart: Implications for obesity

Collagen IV is the major matrix component associated with differentiating adipocytes in adipose tissues, and the understanding of its contribution in adipogenic differentiation could be important for elucidation of mechanisms and processes driving the obesity. Therefore, in the light of our previous findings of differential effects of structural conformation of collagen I matrix on differentiation of bone marrow stromal cells, we investigated whether similar phenomenon occurs on collagen IV matrix in native and denatured structural states. The results of the present study show that native collagen IV is unsupportive of adipogenic differentiation and very little if any adipogenesis occurs on this matrix in the presence of adipogenic stimuli. In sharp contrast to native collagen IV, the same matrix in denatured structural state drives highly efficient adipogenic differentiation suggesting that it might be the major driver of adipogenesis in adipose tissues and that the ratio of native to denatured matrix might regulate the intensity of adipogenesis and possibly underlies the obesity. In contrast to observations that adipogenesis on denatured collagen I (collagen I is the major matrix component in musculoskeletal tissues) is suppressed by stress, adipogenesis on denatured collagen IV appears to be stress-resistant suggesting an explanation for the observed ineffectiveness of physical exercise, i.e. mechanical stress, in the reduction of adipose tissues. The obesity was shown to be associated with overproduction of MMPs and decline in levels of TIMPs. Such a shift in MMP/TIMP balance was considered a consequence of the pathology. In the light of the present study, however, this shift might constitute the primary source of the decease. The findings of the present study suggest strategies for the treatment of obesity, raise significant questions and indicate directions for further experimentation.     

Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process

The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxic shift reprogramming and has glyoxalase activity. We show that substoichiometric concentrations of Hsp31 can abrogate aggregation of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of α-synuclein (αSyn), a target of the chaperone activity of human DJ-1, an Hsp31 homolog. We demonstrate that Hsp31 is able to suppress the in vitro fibrillization or aggregation of αSyn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of αSyn cytoplasmic foci, and yeast cells were rescued from αSyn-generated proteotoxicity upon Hsp31 overexpression. Moreover, we showed that Hsp31 protein levels are increased by H₂O₂, in the diauxic phase of normal growth conditions, and in cells under αSyn-mediated proteotoxic stress. We show that Hsp31 chaperone activity and not the methylglyoxalase activity or the autophagy pathway drives the protective effects. We also demonstrate reduced aggregation of the Sup35 prion domain, PrD-Sup35, as visualized by fluorescent protein fusions. In addition, Hsp31 acts on its substrates prior to the formation of large aggregates because Hsp31 does not mutually localize with prion aggregates, and it prevents the formation of detectable in vitro αSyn fibrils. These studies establish that the protective role of Hsp31 against cellular stress is achieved by chaperone activity that intervenes early in the protein misfolding process and is effective on a wide spectrum of substrate proteins, including αSyn and prion proteins.     

Release of Tensile Strain on Engineered Human Tendon Tissue Disturbs Cell Adhesions,Changes Matrix Architecture,and Induces an Inflammatory Phenotype

Mechanical loading of tendon cells results in an upregulation of mechanotransduction signaling pathways, cell-matrix adhesion and collagen synthesis, but whether unloading removes these responses is unclear. We investigated the response to tension release, with regard to matrix proteins, pro-inflammatory mediators and tendon phenotypic specific molecules, in an in vitro model where tendon-like tissue was engineered from human tendon cells. Tissue sampling was performed 1, 2, 4 and 6 days after surgical de-tensioning of the tendon construct. When tensile stimulus was removed, integrin type collagen receptors showed a contrasting response with a clear drop in integrin subunit α₁₁ mRNA and protein expression, and an increase in α₂ integrin mRNA and protein levels. Further, specific markers for tendon cell differentiation declined and normal tendon architecture was disturbed, whereas pro-inflammatory molecules were upregulated. Stimulation with the cytokine TGF-β1 had distinct effects on some tendon-related genes in both tensioned and de-tensioned tissue. These findings indicate an important role of mechanical loading for cellular and matrix responses in tendon, including that loss of tension leads to a decrease in phenotypical markers for tendon, while expression of pro-inflammatory mediators is induced.     

The Streptococcal Collagen-binding Protein CNE Specifically Interferes with ��V��3-mediated Cellular Interactions with Triple Helical Collagen

Collagen fibers expose distinct domains allowing for specific interactions with other extracellular matrix proteins and cells. To investigate putative collagen domains that govern integrin α_Vβ₃-mediated cellular interactions with native collagen fibers we took advantage of the streptococcal protein CNE that bound native fibrillar collagens. CNE specifically inhibited α_Vβ₃-dependent cell-mediated collagen gel contraction, PDGF BB-induced and α_Vβ₃-mediated adhesion of cells, and binding of fibronectin to native collagen. Using a Toolkit composed of overlapping, 27-residue triple helical segments of collagen type II, two CNE-binding sites present in peptides II-1 and II-44 were identified. These peptides lack the major binding site for collagen-binding β₁ integrins, defined by the peptide GFOGER. Peptide II-44 corresponds to a region of collagen known to bind collagenases, discoidin domain receptor 2, SPARC (osteonectin), and fibronectin. In addition to binding fibronectin, peptide II-44 but not II-1 inhibited α_Vβ₃-mediated collagen gel contraction and, when immobilized on plastic, supported adhesion of cells. Reduction of fibronectin expression by siRNA reduced PDGF BB-induced α_Vβ₃-mediated contraction. Reconstitution of collagen types I and II gels in the presence of CNE reduced collagen fibril diameters and fibril melting temperatures. Our data indicate that contraction proceeded through an indirect mechanism involving binding of cell-produced fibronectin to the collagen fibers. Furthermore, our data show that cell-mediated collagen gel contraction does not directly depend on the process of fibril formation.     

β1 Integrin-mediated collagen gel contraction is stimulated by PDGF

The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent β₁ integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-β1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin β₁-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-β₁ integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the β₁ integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.     

The Hsp70 Homologue Lhs1p Is Involved in a Novel Function of the Yeast Endoplasmic Reticulum, Refolding and Stabilization of Heat-denatured Protein Aggregates

Heat stress is an obvious hazard, and mechanisms to recover from thermal damage, largely unknown as of yet, have evolved in all organisms. We have recently shown that a marker protein in the ER of Saccharomyces cerevisiae, denatured by exposure of cells to 50°C after preconditioning at 37°C, was reactivated by an ATP-dependent machinery, when the cells were returned to physiological temperature 24°C. Here we show that refolding of the marker enzyme Hsp150Δ–β-lactamase, inactivated and aggregated by the 50°C treatment, required a novel ER-located homologue of the Hsp70 family, Lhs1p. In the absence of Lhs1p, Hsp150Δ–β-lactamase failed to be solubilized and reactivated and was slowly degraded. Coimmunoprecipitation experiments suggested that Lhs1p was somehow associated with heat-denatured Hsp150Δ– β-lactamase, whereas no association with native marker protein molecules could be detected. Similar findings were obtained for a natural glycoprotein of S. cerevisiae, pro-carboxypeptidase Y (pro-CPY). Lhs1p had no significant role in folding or secretion of newly synthesized Hsp150Δ–β-lactamase or pro-CPY, suggesting that the machinery repairing heat-damaged proteins may have specific features as compared to chaperones assisting de novo folding. After preconditioning and 50°C treatment, cells lacking Lhs1p remained capable of protein synthesis and secretion for several hours at 24°C, but only 10% were able to form colonies, as compared to wild-type cells. We suggest that Lhs1p is involved in a novel function operating in the yeast ER, refolding and stabilization against proteolysis of heatdenatured protein. Lhs1p may be part of a fundamental heat-resistant survival machinery needed for recovery of yeast cells from severe heat stress.     

Hydrogen Sulfide Alleviates Myocardial Collagen Remodeling in Association with Inhibition of TGF-β/Smad Signaling Pathway in Spontaneously Hypertensive Rats

The study was designed to explore the role and possible mechanisms of hydrogen sulfide (H₂S) in the regulation of myocardial collagen remodeling in spontaneously hypertensive rats (SHRs). We treated nine-week-old male SHRs and age- and sex-matched Wistar–Kyoto rats (WKYs) with NaHS (90 μmol/kg⁻¹·day⁻¹) for 9 wks. At 18 wks, plasma H₂S, tail arterial pressure, morphology of the heart, myocardial ultrastructure and collagen volume fraction (CVF), myocardial expressions of collagen I and III protein and procollagen I and III mRNA, transforming growth factor-β1 (TGF-β1), TGF-β type I receptor (TβR-I), type II receptor (TβR-II), p-Smad2 and 3, matrix metalloproteinase (MMP)-13 and tissue inhibitors of MMP (TIMP)-1 proteins were determined. TGF-β1-stimulated cultured cardiac fibroblasts (CFs) were used to further study the mechanisms. The results showed that compared with WKYs, SHRs showed a reduced plasma H₂S, elevated tail artery pressure and increased myocardial collagen, TGF-β1, TβR-II, p-Smad2 and p-Smad3 expressions. However, NaHS markedly decreased tail artery pressure and inhibited myocardial collagen, TGF-β1, TβR-II, p-Smad2 and p-Smad3 protein expressions, but H₂S had no effect on the expressions of MMP-13 and TIMP-1. Hydralazine reduced blood pressure but had no effect on myocardial collagen, MMP-13 and TIMP-1 expressions and TGF-β1/Smad signaling pathway. H₂S prevented activation of the TGF-β1/Smad signaling pathway and abnormal collagen synthesis in CFs. In conclusion, the results suggested that H₂S could prevent myocardial collagen remodeling in SHR. The mechanism might be associated with inhibition of collagen synthesis via TGF-β1/Smad signaling pathway.     

Characterization of the interaction between alphaCP(2) and the 3'-untranslated region of collagen alpha1(I) mRNA

Activated hepatic stellate cells produce increased type I collagen in hepatic fibrosis. The increase in type I collagen protein results from an increase in mRNA levels that is mainly mediated by increased mRNA stability. Protein–RNA interactions in the 3′-UTR of the collagen α1(I) mRNA correlate with stabilization of the mRNA during hepatic stellate cell activation. A component of the binding complex is αCP₂. Recombinant αCP₂ is sufficient for binding to the 3′-UTR of collagen α1(I). To characterize the binding affinity of and specificity for αCP₂, we performed electrophoretic mobility shift assays using the poly(C)-rich sequence in the 3′-UTR of collagen α1(I) as probe. The binding affinity of αCP₂ for the 3′-UTR sequence is ~2 nM in vitro and the wild-type 3′ sequence binds with high specificity. Furthermore, we demonstrate a system for detecting protein–nucleotide interactions that is suitable for high throughput assays using molecular beacons. Molecular beacons, developed for DNA–DNA hybridization, are oligonucleotides with a fluorophore and quencher brought together by a hairpin sequence. Fluorescence increases when the hairpin is disrupted by binding to an antisense sequence or interaction with a protein. Molecular beacons displayed a similar high affinity for binding to recombinant αCP₂ to the wild-type 3′ sequence, although the kinetics of binding were slower.     

Thermal denaturation of soluble calf-skin collagen

1. Soluble calf-skin collagen has been denatured thermally between 37° and 60° and the component proteins have been separated on carboxymethylcellulose. 2. Four main fractions have been separated; α and β (in the nomenclature in common usage) and two other fractions. (The α and β components are complex owing to the presence of α₁, α₂, β₁ and β₂ parts). 3. Fractions 3 and 4 undergo rapid denaturation between 39° and 40° whereafter fraction 4 remains virtually unchanged even at 60°. 4. That portion of fraction 4 which remains at 60° is thought to be identical with the fraction designated γ by other workers, this fraction being composed of three α-chains in covalent linkage (such bonds are alkali-labile). 5. The equilibrium between α, β and fractions 3 and 4 is apparently reversible since acid-soluble collagen after denaturation at 45° or 60° followed by cooling to 0° for 30min. was found to contain only fraction 4 when chromatographed at 37°.