Regulation Of Collagen Synthesis Research Articles

Bradykinin inhibits high glucose- and growth factor-induced collagen synthesis in mesangial cells through the B2-kinin receptor

Mesangial matrix expansion is an early lesion leading to glomeruloclerosis and chronic renal diseases. A beneficial effect is achieved with angiotensin I-converting enzyme inhibitors (ACEI), which also favor bradykinin (BK) B2 receptor (B2R) activation. To define the underlying mechanism, we hypothesized that B2R activation could be a negative regulator of collagen synthesis in mesangial cells (MC). We investigated the effect of BK on collagen synthesis and signaling in MC. Inflammation was evaluated by intercellular adhesion molecule-1 (ICAM-1) expression. BK inhibited collagen I and IV synthesis stimulated by high glucose, epithelial growth factor (EGF), and transforming growth factor-β (TGF-β) but did not alter ICAM-1. Inhibition of collagen synthesis was B2R but not B1R mediated. PKC or phosphatidylinositol 3-kinase (PI3K) inhibitors mimicked the BK effect. B2R activation inhibited TGF-β- and EGF-induced Erk1/2, Smad2/3, Akt S473, and EGFR phosphorylation. A phosphatase inhibitor prevented BK effects. The in vivo impact of B2R on mesangial matrix expansion was assessed in streptozotocin-diabetic rodents. Deletion of B2R increased mesangial matrix expansion and albuminuria in diabetic mice. In diabetic rats, matrix expansion and albuminuria were prevented by ACEI but not by ACEI and B2R antagonist cotreatment. Consistently, the lowered BK content of diabetic glomeruli was restored by ACEI. In conclusion, deficient B2R activation aggravated mesangial matrix expansion in diabetic rodents whereas B2R activation reduced MC collagen synthesis by a mechanism targeting Erk1/2 and Akt, common pathways activated by EGF and TGF-β. Taken together, the data support the hypothesis of an antifibrosing effect of B2R activation.

Role of interleukin‐33 in sepsis‐induced myocardial dysfunction

The disruption of myocardial extracellular matrix (ECM) protein has been implicated in myocardial dysfunction during sepsis. However, the underlying mechanism(s) are not clear. Interleukin‐ 33 (IL‐33) is a cytokine which can regulate collagen synthesis in various cardiac pathologies. The purpose of the present study is to test whether IL‐33 contributes to the sepsis‐induced myocardial dysfunction through regulation of matrix metalloprotease‐9 (MMP‐ 9).MethodsIn vivo, feces‐induced peritonitis (FIP) in mice and in vitro LPS treatments to isolated cardiomyocytes were used in the study.ResultsIn mice with FIP, myocardial IL‐33 and MMP‐9 expression were increased and myocardial contractility was decreased. Myocardial function in mice with FIP was improved when the mice were treated with soluble ST2 (sST2), a decoy receptor of IL‐33. The in vitro, expression of IL‐33 and MMP‐9 in cardiomyocytes treated with LPS was increased. Addition of sST2 prevented the increase in MMP‐9 expression in LPS treated myocytes.ConclusionOur results indicate IL‐33 plays an important role in mediating sepsis‐induced myocardial dysfunction by regulation of myocyte MMP‐9 expression. (Supported by the Department of National Defense, Canadian Forces Medical Group)

Inhibition of Integrin α2β1 Ameliorates Glomerular Injury

Mesangial cells and podocytes express integrins α1β1 and α2β1, which are the two major collagen receptors that regulate multiple cellular functions, including extracellular matrix homeostasis. Integrin α1β1 protects from glomerular injury by negatively regulating collagen production, but the role of integrin α2β1 in renal injury is unclear. Here, we subjected wild-type and integrin α2-null mice to injury with adriamycin or partial renal ablation. In both of these models, integrin α2-null mice developed significantly less proteinuria and glomerulosclerosis. In addition, selective pharmacological inhibition of integrin α2β1 significantly reduced adriamycin-induced proteinuria, glomerular injury, and collagen deposition in wild-type mice. This inhibitor significantly reduced collagen synthesis in wild-type, but not integrin α2-null, mesangial cells in vitro, demonstrating that its effects are integrin α2β1-dependent. Taken together, these results indicate that integrin α2β1 contributes to glomerular injury by positively regulating collagen synthesis and suggest that its inhibition may be a promising strategy to reduce glomerular injury and proteinuria.

Monoclonal antibody against marinobufagenin reverses cardiac fibrosis in rats with chronic renal failure

Cardiotonic steroids (CTS) are implicated in pathophysiology of uremic cardiomyopathy. In the present study, we tested whether a monoclonal antibody (mAb) against the bufadienolide CTS, marinobufagenin (MBG), alleviates cardiac hypertrophy and fibrosis in partially nephrectomized (PNx) rats. In PNx rats, we compared the effects of 3E9 anti-MBG mAb and of Digibind, an affinity-purified digoxin antibody, on blood pressure and cardiac hypertrophy and fibrosis following 4 weeks after the surgery. In PNx rats, a fourfold elevation in plasma MBG levels was associated with hypertension, increased cardiac levels of carbonylated protein, cardiac hypertrophy, a reduction in cardiac expression of a nuclear transcription factor which is a negative regulator of collagen synthesis, Friend leukemia integration-1 (Fli-1), and an increase in the levels of collagen-1. A single intraperitoneal administration of 3E9 mAb to PNx rats reduced blood pressure by 59 mm Hg for 7 days and produced a significant reduction in cardiac weight and cardiac levels of oxidative stress, an increase in the expression of Fli-1, and a reduction in cardiac fibrosis. The effects of Digibind were similar to those of 3E9 mAb, but were less pronounced. In experimental chronic renal failure, elevated levels of MBG contribute to hypertension and induce cardiac fibrosis via suppression of Fli-1, representing a potential target for therapy.

Fibrosis: is it a coactivator disease?

Fibrosis is an abnormal fibroblast-activation-associated pathological manifestation in injured organs where excessive non-physiological synthesis and accumulation of extracellular matrix (ECM) proteins by activated/differentiated fibroblasts disrupts tissue homeostasis. Like other eukaryotic genes, expression of ECM protein genes not only depends on its gene sequences in the regulatory region but also influenced by non-genetic factors called epigenetic regulators including acetyltransferases, deacetylases, methyltransferases and microRNAs. The acetyltransferase p300 (ATp300), a transcriptional coactivator, is a major player in the epigenetic regulation of genes whose products are involved in cellular growth, proliferation, apoptosis and essential for embryonic development. ATp300 acetylates specific lysine residues in histones and transcription factors (KAT) and as a transcriptional coactivator it forms a bridge between upstream regulatory element binding protein complex and basal transcriptional machinery. Abnormal coactivator activity-associated diseases are known as coactivator diseases. Abnormalities in ATp300 activities in adults are associated with numerous diseases. Here, we review the significant roles of ATp300 in epigenetic regulation of collagen synthesis and deposition in extracellular spaces, matrix remodeling and tissue fibrogenesis. The present day understanding on the distinct role of acetyltransferases, deacetylases, and deacetylase inhibitors on epigenetic regulation of matrix remodeling and fibrosis has also been discussed.

Fibrosis is it a coactivator disease

Fibrosis is an abnormal fibroblast-activation-associated pathological manifestation in injured organs where excessive non-physiological synthesis and accumulation of extracellular matrix (ECM) proteins by activated/differentiated fibroblasts disrupts tissue homeostasis. Like other eukaryotic genes, expression of ECM protein genes not only depends on its gene sequences in the regulatory region but also influenced by non-genetic factors called epigenetic regulators including acetyltransferases, deacetylases, methyltransferases and microRNAs. The acetyltransferase p300 (ATp300), a transcriptional coactivator, is a major player in the epigenetic regulation of genes whose products are involved in cellular growth, proliferation, apoptosis and essential for embryonic development. ATp300 acetylates specific lysine residues in histones and transcription factors (KAT) and as a transcriptional coactivator it forms a bridge between upstream regulatory element binding protein complex and basal transcriptional machinery. Abnormal coactivator activity-associated diseases are known as coactivator diseases. Abnormalities in ATp300 activities in adults are associated with numerous diseases. Here, we review the significant roles of ATp300 in epigenetic regulation of collagen synthesis and deposition in extracellular spaces, matrix remodeling and tissue fibrogenesis. The present day understanding on the distinct role of acetyltransferases, deacetylases, and deacetylase inhibitors on epigenetic regulation of matrix remodeling and fibrosis has also been discussed.

Abstract P129: G Protein--Coupled Receptor Kinase-2 (GRK2) Is a Novel Regulator of Collagen Synthesis in Adult Human Cardiac Fibroblasts

Cardiac fibroblasts (CF) make up 70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. The objective of this study was to investigate the potential role of GRK2 in modulating CF transformation to myofibroblasts and collagen synthesis in adult human CF isolated from normal and failing left ventricles. CF isolated from failing ventricles showed a significant increase in expression of collagen I, III and VI compared with controls. α-SMA was increased 2-fold over controls, consistent with CF transformation to myofibroblasts. Baseline collagen synthesis was elevated 2-fold in failing CF and was not inhibited by isoproterenol (ISO)-stimulation in contrast to normal controls. β-adrenergic receptor (β-AR) signaling was markedly uncoupled in failing CF as assessed by basal and ISO-stimulated cAMP production. The primary mechanism appears to be a 2.5-fold increase in GRK2 activity as GRK2 phosphorylates and uncouples agonist-occupied β-ARs. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following ISO stimulation. In contrast, siRNA-mediated knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater β-agonist-mediated inhibition of basal and TGFβ-stimulated collagen synthesis versus control. Inhibition of GRK2 activity by adenoviral-mediated βARKct expression or GRK2 knockdown in failing CF led to a significant decline in collagen and α-SMA expression. GRK2 inhibition restored β-AR signaling and ISO-stimulated inhibition of collagen synthesis and also significantly decreased collagen synthesis in response to TGFβ stimulation. In conclusion, GRK2 appears to play a significant role in regulating CF transformation and collagen synthesis in adult human CF and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.

Myocardin-related Transcription Factor-A Complexes Activate Type I Collagen Expression in Lung Fibroblasts

Pulmonary fibrosis is characterized by the excessive deposition of a collagen-rich extracellular matrix. The accumulation of collagen within the lung interstitium leads to impaired respiratory function. Furthermore, smooth muscle actin-positive myofibroblasts within the fibrotic lung contribute to disease progression. Because collagen and smooth muscle cell α-actin are coordinately expressed in the setting of fibrosis, the hypothesis was tested that specific transcriptional regulators of the myocardin family might also regulate collagen gene expression in myofibroblasts. Myocardin-related transcription factors (MRTFs), through their interaction with the serum-response factor (SRF) on CArG box regulatory elements (CC(A/T)6GG), are important regulators of myofibroblast differentiation. MRTF-A transactivated type I collagen gene reporters as much as 100-fold in lung myofibroblasts. Loss of functional MRTF-A using either a dominant negative MRTF-A isoform, shRNA targeting MRTF-A, or genetic deletion of MRTF-A in lung fibroblasts significantly disrupted type I collagen synthesis relative to controls. Analysis of the COL1A2 proximal promoter revealed a noncanonical CArG box (CCAAACTTGG), flanked by several Sp1 sites important for MRTF-A activation. Chromatin immunoprecipitation experiments confirmed the co-localization of MRTF-A, SRF, and Sp1 bound to the same region of the COL1A2 promoter. Mutagenesis of either the noncanonical CArG box or the Sp1 sites significantly disrupted MRTF-A activation of COL1A2. Together, our findings show that MRTF-A is an important regulator of collagen synthesis in lung fibroblasts and exhibits a dependence on both SRF and Sp1 function to enhance collagen expression.

Cyclic nucleotide phosphodiesterase 1A: a key regulator of cardiac fibroblast activation and extracellular matrix remodeling in the heart

Cardiac fibroblasts become activated and differentiate to smooth muscle-like myofibroblasts in response to hypertension and myocardial infarction (MI), resulting in extracellular matrix (ECM) remodeling, scar formation and impaired cardiac function. cAMP and cGMP-dependent signaling have been implicated in cardiac fibroblast activation and ECM synthesis. Dysregulation of cyclic nucleotide phosphodiesterase (PDE) activity/expression is also associated with various diseases and several PDE inhibitors are currently available or in development for treating these pathological conditions. The objective of this study is to define and characterize the specific PDE isoform that is altered during cardiac fibroblast activation and functionally important for regulating myofibroblast activation and ECM synthesis. We have found that Ca(2+)/calmodulin-stimulated PDE1A isoform is specifically induced in activated cardiac myofibroblasts stimulated by Ang II and TGF-β in vitro as well as in vivo within fibrotic regions of mouse, rat, and human diseased hearts. Inhibition of PDE1A function via PDE1-selective inhibitor or PDE1A shRNA significantly reduced Ang II or TGF-β-induced myofibroblast activation, ECM synthesis, and pro-fibrotic gene expression in rat cardiac fibroblasts. Moreover, the PDE1 inhibitor attenuated isoproterenol-induced interstitial fibrosis in mice. Mechanistic studies revealed that PDE1A modulates unique pools of cAMP and cGMP, predominantly in perinuclear and nuclear regions of cardiac fibroblasts. Further, both cAMP-Epac-Rap1 and cGMP-PKG signaling was involved in PDE1A-mediated regulation of collagen synthesis. These results suggest that induction of PDE1A plays a critical role in cardiac fibroblast activation and cardiac fibrosis, and targeting PDE1A may lead to regression of the adverse cardiac remodeling associated with various cardiac diseases.

Relaxin Regulates Myofibroblast Contractility and Protects against Lung Fibrosis

Myofibroblasts are specialized contractile cells that participate in tissue fibrosis and remodeling, including idiopathic pulmonary fibrosis (IPF). Mechanotransduction, a process by which mechanical stimuli are converted into biochemical signals, regulates myofibroblast differentiation. Relaxin is a peptide hormone that mediates antifibrotic effects through regulation of collagen synthesis and turnover. In this study, we demonstrate enhanced myofibroblast contraction in bleomycin-induced lung fibrosis in mice and in fibroblastic foci of human subjects with IPF, using phosphorylation of the regulatory myosin light chain (MLC(20)) as a biomarker of in vivo cellular contractility. Compared with wild-type mice, relaxin knockout mice express higher lung levels of phospho-MLC(20) and develop more severe bleomycin-induced lung fibrosis. Exogenous relaxin inhibits MLC(20) phosphorylation and bleomycin-induced lung fibrosis in both relaxin knockout and wild-type mice. Ex vivo studies of IPF lung myofibroblasts demonstrate decreases in MLC(20) phosphorylation and reduced contractility in response to relaxin. Characterization of the signaling pathway reveals that relaxin regulates MLC(20) dephosphorylation and lung myofibroblast contraction by inactivating RhoA/Rho-associated protein kinase through a nitric oxide/cGMP/protein kinase G-dependent mechanism. These studies identify a novel antifibrotic role of relaxin involving the inhibition of the contractile phenotype of lung myofibroblasts and suggest that targeting myofibroblast contractility with relaxin-like peptides may be of therapeutic benefit in the treatment of fibrotic lung disease.

G Protein-coupled Receptor Kinase-2 Is a Novel Regulator of Collagen Synthesis in Adult Human Cardiac Fibroblasts

Cardiac fibroblasts (CF) make up 60-70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction and the transition to heart failure. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. We investigated the potential role of G protein-coupled receptor kinase-2 (GRK2) in modulating collagen synthesis by adult human CF isolated from normal and failing left ventricles. Baseline collagen synthesis was elevated in failing CF and was not inhibited by β-agonist stimulation in contrast to normal controls. β-adrenergic receptor (β-AR) signaling was markedly uncoupled in the failing CF, and expression and activity of GRK2 were increased 3-fold. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following β-agonist stimulation. In contrast, knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater β-agonist-mediated inhibition of basal and TGFβ-stimulated collagen synthesis versus control. Inhibition of GRK2 activity in failing CF by expression of the GRK2 inhibitor, GRK2ct, or siRNA-mediated knockdown restored β-agonist-stimulated inhibition of collagen synthesis and decreased collagen synthesis in response to TGFβ stimulation. GRK2 appears to play a significant role in regulating collagen synthesis in adult human CF, and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.

Angiotensin II up‐regulates the expression of scleraxis in cardiac myofibroblasts isolated from the site of myocardial infarction

Scleraxis (scx) is a basic helix-loop-helix transcription factor involved in TGF-β1 induced collagen expression and fibrosis. Recent studies have implicated scx to play a role in regulating collagen synthesis in differentiated fibroblast. However, scx role in myocardial infarction (MI) and cardiac remodeling is unknown. Moreover, Angiotensin II (AngII) is a known mediator of collagen expression in MI. Therefore, it's interesting to examine the effects of AngII on scx using cardiac myofibroblasts (myoFb) isolated from the site of 28-day post-MI rat heart; these cells are implicated in adverse remodeling and fibrosis at the site of MI. In the present study, we used primary cultures of adult rat cardiac myoFb isolated from the site of 28-day post-MI heart and confluent myoFb were treated with (10−7 M) AngII, AngII + Losartan (AngII type 1 [AT1] receptor blocker) and an untreated control group. Our results from both western blot and qRT-PCR illustrated significant up regulation (p<0.05) of scx expression at gene and protein levels in the presence of AngII. Losartan treatment (1μM) abrogated the AngII induced expression of scx in cardiac myoFb in AngII + Losartan group. The data presented demonstrates that AngII effects are mediated by AT1 receptor and AngII modulates scx expression either directly or indirectly via TGF-β1.

Interaction of scleraxis and Smad‐dependent cardiac collagen expression

Deposition of type I collagen is a hallmark of cardiac fibrosis. We have shown that the transcription factor scleraxis (Scx) is expressed in cardiac fibroblasts and is sufficient to regulate human collagen Iα2 (COLIα2) expression. Our objective was to study Scx interaction with the fibrotic Smad signalling pathway. We identified specific E box sites within the COLIα2 proximal promoter mediating transactivation by Scx. TGF-β1 augmented transactivation of the promoter by Scx, suggesting an additive effect of Scx with TGF-β1 signalling pathways. Inhibitory Smad7 partially blunted Scx-mediated COLIα2 promoter activation. Receptor Smad3 up-regulated whereas Smad7 down-regulated Scx expression in primary rat cardiac fibroblasts, suggesting cross-talk between Scx and Smad-mediated COLIα2 expression mechanisms. A dominant-negative Scx mutant abrogated COLIα2 promoter transactivation by TGF-β1, suggesting that interference with Scx function is sufficient to block pro-fibrotic signalling. We noted elevated Scx expression in the pressure-overloaded murine heart following thoracic aortic banding concomitant with increased type I collagen expression. These results support our hypothesis that Scx regulates collagen synthesis and hence may be an important novel target for developing anti-fibrotic therapies. Supported by Canadian Institutes of Health Research & St. Boniface Hospital Research Foundation.

Proteins mediating collagen biosynthesis and accumulation in arterial repair: novel targets for anti-restenosis therapy

Events contributing to restenosis after coronary interventions include platelet aggregation, inflammatory cell infiltration, growth factor release, and accumulation of smooth muscle cells (SMCs) and extracellular matrix (ECM). The ECM is composed of various collagen subtypes and proteoglycans and over time constitutes the major component of the mature restenotic plaque. The pathophysiology of collagen accumulation in the ECM during arterial restenosis is reviewed. Factors regulating collagen synthesis and degradation, including various cytokines and growth factors involved in the process, may be targets for therapies aimed at prevention of in-stent restenosis.

4-phenylbutyric Acid Regulates Collagen Synthesis and Secretion Induced by High Concentrations of Glucose in Human Gingival Fibroblasts

High glucose leads to physio/pathological alterations in diabetes patients. We investigated collagen production in human gingival cells that were cultured in high concentrations of glucose. Collagen synthesis and secretion were increased when the cells were exposed to high concentrations of glucose. We examined endoplasmic reticulum (ER) stress response because glucose metabolism is related to ER functional status. An ER stress response including the expression of glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), inositol requiring enzyme alpha (IRE-1α) and phosphoreukaryotic initiation factor alpha (p-eIF-2α) was activated in the presence of high glucose. Activating transcription factor 4 (ATF-4), a downstream protein of p-eIF-2α as well as a transcription factor for collagen, was also phosphorylated and translocalized into the nucleus. The chemical chaperone 4-PBA inhibited the ER stress response and ATF-4 phosphorylation as well as nuclear translocation. Our results suggest that high concentrations of glucose-induced collagen are linked to ER stress and the associated phosphorylation and nuclear translocation of ATF-4.

Signaling through the Na/K-ATPase: implications for cardiac fibrosis

the cardiotonic effects of digitalis have been exploited in clinical practice for many years. However, while the existence of endogenous digitalis-like substances (also called cardiotonic steroids) has been accepted for some time, their exact chemical nature has been only recently demonstrated ([1][

Combined use of bFGF and GDF-5 enhances the healing of medial collateral ligament injury

Basic fibroblast growth factor (bFGF) and growth and differentiation factor (GDF)-5 stimulate the healing of medial collateral ligament (MCL) injury. However, the effect of isolated and combined use of bFGF/GDF-5 remains still unclear. We investigated cellular proliferation and migration responding to bFGF/GDF-5 using rabbit MCL fibroblasts. Rabbit MCL injury was treated by bFGF and/or GDF-5 with peptide hydrogels. Gene expression and deposition of collagens in healing tissues were evaluated. bFGF/GDF-5 treatment additively enhanced cell proliferation and migration. bFGF/GDF-5 hydrogels stimulated Col1a1 expression without increasing Col3a1 expression. Combined use of bFGF/GDF-5 stimulated type I collagen deposition and the reorganization of fiber alignment, and induced better morphology of fibroblasts in healing MCLs. Our study indicates that combined use of bFGF/GDF-5 might enhance MCL healing by increasing proliferation and migration of MCL fibroblasts, and by regulating collagen synthesis and connective fiber alignment.

Expression in SPARC-null mice of collagen type I lacking the globular domain of the α1(I) N-propeptide results in abdominal hernias and loss of dermal collagen

The sequence encoding the N-propeptide of collagen I is characterized by significant conservation of amino acids across species; however, the function of the N-propeptide remains poorly defined. Studies in vitro have suggested that one activity of this propeptide might be to act as a feedback inhibitor of collagen I synthesis. To determine whether the N-propeptide contributed to decreased collagen content in SPARC-null mice, mice carrying a deletion of exon 2, which encodes the globular domain of the N-propeptide of collagen I, were crossed to SPARC-null animals. Mice lacking SPARC and expressing collagen I without the globular domain of the N-propeptide were viable and fertile. However, a significant number of animals developed abdominal hernias within the first 2months of life with an approximate 20% penetrance (~35% of males). The dermis of SPARC-null/exon 2-deleted mice was thinner and contained fewer large collagen fibers in comparison with wild-type or in either single transgenic animal. The average collagen fibril diameter of exon 2-deleted mice did not significantly differ from wild-type mice (WT: 87.9nm versus exon 2-deleted: 88.2nm), whereas SPARC-null/exon 2-deleted fibrils were smaller than that of SPARC-null dermis (SPARC-null: 60.2nm, SPARC-null/exon 2-deleted: 40.8nm). As measured by hydroxyproline analysis, double transgenic skin biopsies contained significantly less collagen than those of wild-type, those of exon 2-deleted, and those of SPARC-null biopsies. Acetic acid extraction of collagen from skin biopsies revealed an increase in the proportion of soluble collagen in the SPARC-null/exon 2-deleted mice. These results support a function of the N-propeptide of collagen I in facilitating incorporation and stabilization of collagen I into the insoluble ECM and argue against a primary function of the N-propeptide as a negative regulator of collagen synthesis.

T3 affects expression of collagen I and collagen cross-linking in bone cell cultures

Thyroid hormones (T3,T4) have a broad range of effects on bone, however, its role in determining the quality of bone matrix is poorly understood. In-vitro, the immortalized mouse osteoblast-like cell line MC3T3-E1 forms a tissue like structure, consisting of several cell layers, whose formation is affected by T3 significantly. In this culture system, we investigated the effects of T3 on cell multiplication, collagen synthesis, expression of genes related to the collagen cross-linking process and on the formation of cross-links.T3 compared to controls modulated cell multiplication, up-regulated collagen synthesis time and dose dependently, and stimulated protein synthesis. T3 increased mRNA expressions of procollagen-lysine-1,2-oxoglutarate 5-dioxygenase 2 (Plod2) and of lysyloxidase (Lox), both genes involved in post-translational modification of collagen. Moreover, it stimulated mRNA expression of bone morphogenetic protein 1 (Bmp1), the processing enzyme of the lysyloxidase-precursor and of procollagen. An increase in the collagen cross-link-ratio Pyr/deDHLNL indicates, that T3 modulated cross-link maturation in the MC3T3-E1 culture system. These results demonstrate that T3 directly regulates collagen synthesis and collagen cross-linking by up-regulating gene expression of the specific cross-link related enzymes, and underlines the importance of a well-balanced concentration of thyroid hormones for maintenance of bone quality.

A protocol for enrichment of CD34+ stromal cell fraction through human skin disaggregation and magnetic separation

A protocol for enrichment of CD34+ stromal cell fraction through human skin disaggregation and magnetic separation