RESUMEN
Normalization of secretory activity and differentiation status of mesenchymal cells, including fibroblasts, is an important biomedical problem. One of the possible solutions is modulation of unfolded protein response (UPR) activated during fibroblast differentiation. Here, we investigated the effect of phytohormones on the secretory activity and differentiation of cultured human skin fibroblasts. Based on the analysis of expression of genes encoding UPR markers, abscisic acid (ABA) upregulated expression of the GRP78 and ATF4 genes, while gibberellic acid (GA) upregulated expression of CHOP. Evaluation of the biosynthetic activity of fibroblasts showed that ABA promoted secretion and synthesis of procollagen I and synthesis of fibronectin, as well as the total production of collagen and non-collagen proteins of the extracellular matrix (ECM). ABA also stimulated the synthesis of smooth muscle actin α (α-SMA), which is the marker of myofibroblasts, and increased the number of myofibroblasts in the cell population. On the contrary, GA increased the level of fibronectin secretion, but reduced procollagen I synthesis and the total production of the ECM collagen proteins. GA downregulated the synthesis of α-SMA and decreased the number of myofibroblasts in the cell population. Our results suggest that phytohormones modulate the biosynthetic activity of fibroblasts and affect their differentiation status.
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Fibronectinas , Reguladores del Crecimiento de las Plantas , Humanos , Fibronectinas/genética , Fibronectinas/metabolismo , Fibronectinas/farmacología , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Procolágeno/genética , Procolágeno/metabolismo , Procolágeno/farmacología , Células Cultivadas , Fibroblastos/metabolismo , Miofibroblastos/metabolismo , Diferenciación Celular , Colágeno , Proteínas de la Matriz Extracelular/metabolismo , Actinas/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
Background: The relationship between pelvic organ prolapse (POP), an aging-related disease, and the senescence-related protein mitofusin 2 (Mfn2) has rarely been studied. The aim of the present study was to explore the therapeutic effects of the downregulation of Mfn2 expression by stem cells on POP through animal experiments. Methods: First, a rat POP model was constructed by ovariectomy and traction. The rats in the non-pelvic organ prolapse (NPOP) and POP groups were divided into four groups for negative controls (N1−N4, N1: NPOP-normal saline; N2: NPOP-untransfected stem cells; N3: NPOP-short hairpin negative control (NPOP-sh-NC); N4: NPOP-short hairpin-Mfn2 (NPOP-sh-Mfn2)), and four groups for prolapse (P1−P4, P1: POP-normal saline; P2: POP-untransfected stem cells; P3: POP-sh-NC; P4: POP-sh-Mfn2), respectively. Stem cells were then cultured and isolated. The expression of Mfn2 was inhibited by lentivirus transfection, and the stem cells were injected into the uterosacral ligament of the rats in each group. The expression levels of Mfn2 and procollagen 1A1/1A2/3A1 in the uterosacral ligaments of the rats were observed at 0, 7, 14, and 21 days after injection. Results: Compared to the rats in the NPOP group, the POP rats had significant prolapse. The Mfn2 expression in the uterosacral ligaments of the POP rats was significantly increased (p < 0.05, all), and the expression of procollagen 1A1/1A2/3A1 was significantly decreased (p < 0.001, all). The POP rat model maintained the same trend after 21 days (without stem cell injection). At day 14, compared to the rats in the N1 group, the Mfn2 expression in the uterosacral ligament of the rats in the N4 group was significantly decreased (p < 0.05, all), and the expression of procollagens was significantly increased (p < 0.05, all). Similarly, compared to the rats in the P1 group, the Mfn2 expression in the uterosacral ligament of the rats in the P4 group was significantly decreased (p < 0.05, all), and the expression of procollagens was significantly increased (p < 0.05, all). Similarly, on day 21, the Mfn2 mRNA and protein expression in the uterosacral ligament of the POP and NPOP rats was significantly decreased (p < 0.05, all), and the expression of procollagens was significantly increased (p < 0.05, all) in the rats in the sh-Mfn2 group (N4, P4) compared to the rats in the saline group (N1, P1). Conclusions: The downregulation of Mfn2 expression by stem cells decreased the expression of Mfn2 and increased the expression of procollagen1A1/1A2/3A1 in the uterosacral ligament of the POP rats; this effect was significant 14−21 days after the injection. Thus, Mfn2 may be a new target for POP control.
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GTP Fosfohidrolasas/metabolismo , Células Madre Mesenquimatosas , Proteínas Mitocondriales/metabolismo , Prolapso de Órgano Pélvico , Animales , Regulación hacia Abajo , Femenino , Hidrolasas/genética , Ligamentos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Prolapso de Órgano Pélvico/genética , Prolapso de Órgano Pélvico/metabolismo , Prolapso de Órgano Pélvico/terapia , Posmenopausia , Procolágeno/genética , Procolágeno/metabolismo , Ratas , Solución Salina/metabolismoRESUMEN
Bone morphogenetic protein 1 (BMP1) belongs to the astacin/BMP1/tolloid-like family of zinc metalloproteinases, which play a fundamental role in the development and formation of extracellular matrix (ECM). BMP1 mediates the cleavage of carboxyl terminal (C-term) propeptides from procollagens, a crucial step in fibrillar collagen fiber formation. Blocking BMP1 by small molecule or antibody inhibitors has been linked to anti-fibrotic activity in the preclinical models of skin, kidney and liver fibrosis. Therefore, we reason that BMP1 may be important for the pathogenesis of lung fibrosis and BMP1 could be a potential therapeutic target for progressive fibrotic disease such as idiopathic pulmonary fibrosis (IPF). Here, we observed the increased expression of BMP1 in both human IPF lungs and mouse fibrotic lungs induced by bleomycin. Furthermore, we developed an inducible Bmp1 conditional knockout (cKO) mouse strain. We found that Bmp1 deletion does not protect mice from lung fibrosis triggered by bleomycin. Moreover, we found no significant impact of BMP1 deficiency upon C-term propeptide of type I procollagen (CICP) production in the fibrotic mouse lungs. Based on these results, we propose that BMP1 is not required for lung fibrosis in mice and BMP1 may not be considered a candidate therapeutic target for IPF.
Asunto(s)
Proteína Morfogenética Ósea 1 , Fibrosis Pulmonar Idiopática , Animales , Bleomicina/metabolismo , Proteína Morfogenética Ósea 1/genética , Proteína Morfogenética Ósea 1/metabolismo , Matriz Extracelular/metabolismo , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Ratones , Procolágeno/genéticaRESUMEN
Factors promoting fibroblast proliferation and collagen synthesis can subsequently enhance wound healing. This study aimed to assess the effect of 810 and 940 nm diode laser on fibroblast proliferation and procollagen gene expression. In this study, human gingival fibroblasts were cultured in Dulbecco's modified Eagle's medium and underwent 810 and 940 nm diode laser irradiation once, twice, thrice and four times at 1, 3, 5 and 7 days after culture. The methyl thiazolyl tetrazolium assay was performed to assess the proliferation while the real-time polymerase chain reaction was performed to assess the expression of procollagen gene at the mRNA level. We applied two-way ANOVA and Tukey's test for analysis. Wavelength had no significant effect on the proliferation of gingival fibroblasts, but increasing the number of irradiation sessions of both wavelengths increased the proliferation of human gingival fibroblasts. Significant differences were noted in the number of human gingival fibroblasts between groups irradiated 1 and 4 and also 2 and 4 times. Procollagen gene was well expressed in all groups but its expression was significantly higher in 940 nm laser group after four irradiation cycles. Four times radiation of 940 nm laser seems to be more effective than all others.
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Láseres de Semiconductores , Procolágeno , Humanos , Procolágeno/genética , Procolágeno/metabolismo , Encía , Fibroblastos/efectos de la radiación , Proliferación Celular/efectos de la radiación , Células CultivadasRESUMEN
BACKGROUND: Luffa cylindrica stem sap (LuCS) has been ethnopharmacologically used as a cosmetic ingredients to improve the facial condition in Asians, but there is no scientific proof about the advantages of LuCS as a supplement for skin elasticity inducer. PURPOSE: Presently, we have validated the beneficial effect of LuCS in human preadipocyte and fibroblast. METHODS: In vitro activities of LuCS on expression of cellular elastin and collagen type I were validated using Western blot analysis in human fibroblasts. Effect of LuCS on preadipocyte development was performed using MDI medium containing isobutyl-methylxanthine, dexamethasone, and insulin and then evaluated using oil red O staining. RESULTS: Treatment of LuCS stimulated the expression of cellular elastin and type I procollagen in human skin fibroblasts. Exposure to LuCS induced lipid accumulation of preadipocytes via activation of CEBP/α signaling pathway in preadipocytes. Expression of collagen I, elastin, or CEBP/α mRNA was decreased by age. 3-bromo-3-methylisoxazol-5-amine enhanced the synthesis of cellular lipid in preadipocytes. CONCLUSIONS: Collectively, these results suggest the rationale of LuCS treatment in enhancing the skin condition.
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Adipocitos/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Luffa/química , Extractos Vegetales/farmacología , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Elastina/genética , Elastina/metabolismo , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , Extractos Vegetales/química , Procolágeno/genética , Procolágeno/metabolismoRESUMEN
SEC24 is mainly involved in cargo sorting during COPII vesicle assembly. There are four SEC24 paralogs (A-D) in vertebrates, which are classified into two subgroups (SEC24A/B and SEC24C/D). Pathological mutations in SEC24D cause osteogenesis imperfecta with craniofacial dysplasia in humans. sec24d mutant fish also recapitulate the phenotypes. Consistent with the skeletal phenotypes, the secretion of collagen was severely defective in mutant fish, emphasizing the importance of SEC24D in collagen secretion. However, SEC24D patient-derived fibroblasts show only a mild secretion phenotype, suggesting tissue-specificity in the secretion process. Using Sec24d KO mice and cultured cells, we show that SEC24A and SEC24B also contribute to endoplasmic reticulum (ER) export of procollagen. In contrast, fibronectin 1 requires either SEC24C or SEC24D for ER export. On the basis of our results, we propose that procollagen interacts with multiple SEC24 paralogs for efficient export from the ER, and that this is the basis for tissue-specific phenotypes resulting from SEC24 paralog deficiency.
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Procolágeno , Proteínas de Transporte Vesicular , Animales , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Retículo Endoplásmico/metabolismo , Ratones , Fenotipo , Procolágeno/genética , Procolágeno/metabolismo , Transporte de Proteínas , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Hepatic fibrosis is driven by deposition of matrix proteins following liver injury. Hepatic stellate cells (HSCs) drive fibrogenesis, producing matrix proteins, including procollagen I, which matures into collagen I following secretion. Disrupting intracellular procollagen processing and trafficking causes endoplasmic reticulum stress and stress-induced HSC apoptosis and thus is an attractive antifibrotic strategy. We designed an immunofluorescence-based small interfering RNA (siRNA) screen to identify procollagen I trafficking regulators, hypothesizing that these proteins could serve as antifibrotic targets. A targeted siRNA screen was performed using immunofluorescence to detect changes in intracellular procollagen I. Tumor necrosis factor receptor associated factor 2 and noncatalytic region of tyrosine kinase-interacting kinase (TNIK) was identified and interrogated in vitro and in vivo using the TNIK kinase inhibitor NCB-0846 or RNA interference-mediated knockdown. Our siRNA screen identified nine genes whose knockdown promoted procollagen I retention, including the serine/threonine kinase TNIK. Genetic deletion or pharmacologic inhibition of TNIK through the small molecule inhibitor NCB-0846 disrupted procollagen I trafficking and secretion without impacting procollagen I expression. To investigate the role of TNIK in liver fibrogenesis, we analyzed human and murine livers, finding elevated TNIK expression in human cirrhotic livers and increased TNIK expression and kinase activity in both fibrotic mouse livers and activated primary human HSCs. Finally, we tested whether inhibition of TNIK kinase activity could limit fibrogenesis in vivo. Mice receiving NCB-0846 displayed reduced CCl4 -induced fibrogenesis compared to CCl4 alone, although α-smooth muscle actin levels were unaltered. Conclusions: Our siRNA screen effectively identified TNIK as a key kinase involved in procollagen I trafficking in vitro and hepatic fibrogenesis in vivo.
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Procolágeno , Proteínas Serina-Treonina Quinasas , Animales , Hígado/metabolismo , Ratones , Procolágeno/genética , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , Factor 2 Asociado a Receptor de TNF/metabolismoRESUMEN
Systemic sclerosis (SSc) is a rare autoimmune connective tissue disease characterized by a microangiopathy and fibrosis of the skin and internal organs. No treatment has been proved to be efficient in case of early or advanced SSc to prevent or reduce fibrosis. There are strong arguments for a key role of topo-I in the pathogenesis of diffuse SSc. Irinotecan, a semisynthetic derivative of Camptothecin, specifically target topo-I. This study was undertaken to evaluate the effects of noncytotoxic doses of irinotecan or its active metabolite SN38 on collagen production in SSc fibroblasts. Dermal fibroblasts from 4 patients with SSc and 2 healthy donors were cultured in the presence or absence of irinotecan or SN38. Procollagen I release was determined by ELISA and expression of a panel of genes involved in fibrosis was evaluated by qRT-PCR. Subcytotoxic doses of irinotecan and SN38 caused a significant and dose-dependent decrease of the procollagen I production in dermal fibroblasts from SSc patients, respectively - 48 ± 3%, p < 0.0001 and - 37 ± 6.2%, p = 0.0097. Both irinotecan and SN38 led to a global downregulation of genes involved in fibrosis such as COL1A1, COL1A2, MMP1 and ACTA2 in dermal fibroblasts from SSc patients (respectively - 27; - 20.5; - 30.2 and - 30% for irinotecan and - 61; - 55; - 50 and - 54% for SN38). SN38 increased significantly CCL2 mRNA level (+ 163%). The inhibitory effect of irinotecan and its active metabolite SN38 on collagen production by SSc fibroblasts, which occurs through regulating the levels of expression of genes mRNA, suggests that topoisomerase I inhibitors may be effective in limiting fibrosis in such patients.
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Fibroblastos/efectos de los fármacos , Irinotecán/farmacología , Procolágeno/genética , Esclerodermia Sistémica/genética , Inhibidores de Topoisomerasa I/farmacología , Actinas/genética , Actinas/metabolismo , Estudios de Casos y Controles , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Colágeno Tipo I/antagonistas & inhibidores , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I/antagonistas & inhibidores , Cadena alfa 1 del Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Irinotecán/análogos & derivados , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Cultivo Primario de Células , Procolágeno/antagonistas & inhibidores , Procolágeno/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Piel/metabolismo , Piel/patologíaRESUMEN
PURPOSE: Infantile Caffey disease is a rare disorder characterized by acute inflammation with subperiosteal new bone formation, associated with fever, pain, and swelling of the overlying soft tissue. Symptoms arise within the first weeks after birth and spontaneously resolve before the age of two years. Many, but not all, affected individuals carry the heterozygous pathogenic COL1A1 variant (c.3040C>T, p.(Arg1014Cys)). METHODS: We sequenced COL1A1 in 28 families with a suspicion of Caffey disease and performed ultrastructural, immunocytochemical, and biochemical collagen studies on patient skin biopsies. RESULTS: We identified the p.(Arg1014Cys) variant in 23 families and discovered a novel heterozygous pathogenic COL1A1 variant (c.2752C>T, p.(Arg918Cys)) in five. Both arginine to cysteine substitutions are located in the triple helical domain of the proα1(I) procollagen chain. Dermal fibroblasts (one patient with p.(Arg1014Cys) and one with p.(Arg918Cys)) produced molecules with disulfide-linked proα1(I) chains, which were secreted only with p.(Arg1014Cys). No intracellular accumulation of type I procollagen was detected. The dermis revealed mild ultrastructural abnormalities in collagen fibril diameter and packing. CONCLUSION: The discovery of this novel pathogenic variant expands the limited spectrum of arginine to cysteine substitutions in type I procollagen. Furthermore, it confirms allelic heterogeneity in Caffey disease and impacts its molecular confirmation.
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Cadena alfa 1 del Colágeno Tipo I/genética , Cisteína , Hiperostosis Cortical Congénita , Arginina/genética , Preescolar , Colágeno Tipo I , Cisteína/genética , Humanos , Mutación , Procolágeno/genéticaRESUMEN
Efficient degradation of by-products of protein biogenesis maintains cellular fitness. Strikingly, the major biosynthetic compartment in eukaryotic cells, the endoplasmic reticulum (ER), lacks degradative machineries. Misfolded proteins in the ER are translocated to the cytosol for proteasomal degradation via ER-associated degradation (ERAD). Alternatively, they are segregated in ER subdomains that are shed from the biosynthetic compartment and are delivered to endolysosomes under control of ER-phagy receptors for ER-to-lysosome-associated degradation (ERLAD). Demannosylation of N-linked oligosaccharides targets terminally misfolded proteins for ERAD. How misfolded proteins are eventually marked for ERLAD is not known. Here, we show for ATZ and mutant Pro-collagen that cycles of de-/re-glucosylation of selected N-glycans and persistent association with Calnexin (CNX) are required and sufficient to mark ERAD-resistant misfolded proteins for FAM134B-driven lysosomal delivery. In summary, we show that mannose and glucose processing of N-glycans are triggering events that target misfolded proteins in the ER to proteasomal (ERAD) and lysosomal (ERLAD) clearance, respectively, regulating protein quality control in eukaryotic cells.
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Degradación Asociada con el Retículo Endoplásmico/fisiología , Lisosomas/metabolismo , Polisacáridos/metabolismo , Animales , Calnexina/genética , Calnexina/metabolismo , Fibroblastos/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Humanos , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ratones , Oligosacáridos/metabolismo , Procolágeno/genética , Procolágeno/metabolismo , Pliegue de Proteína , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismoRESUMEN
Osteogenesis Imperfecta (OI) comprises a heterogeneous group of patients who share bone fragility and deformities as the main characteristics, albeit with different degrees of severity. Phenotypic variation also exists in other connective tissue aspects of the disease, complicating disease classification and disease course prediction. Although collagen type I defects are long established as the primary cause of the bone pathology, we are still far from comprehending the complete mechanism. In the last years, the advent of next generation sequencing has triggered the discovery of many new genetic causes for OI, helping to draw its molecular landscape. It has become clear that, in addition to collagen type I genes, OI can be caused by multiple proteins connected to different parts of collagen biosynthesis. The production of collagen entails a complex process, starting from the production of the collagen Iα1 and collagen Iα2 chains in the endoplasmic reticulum, during and after which procollagen is subjected to a plethora of posttranslational modifications by chaperones. After reaching the Golgi organelle, procollagen is destined to the extracellular matrix where it forms collagen fibrils. Recently discovered mutations in components of the retrograde transport of chaperones highlight its emerging role as critical contributor of OI development. This review offers an overview of collagen regulation in the context of recent gene discoveries, emphasizing the significance of transport disruptions in the OI mechanism. We aim to motivate exploration of skeletal fragility in OI from the perspective of these pathways to identify regulatory points which can hint to therapeutic targets.
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Huesos/metabolismo , Colágeno Tipo I/biosíntesis , Osteoblastos/metabolismo , Osteogénesis Imperfecta/metabolismo , Procolágeno/biosíntesis , Procesamiento Proteico-Postraduccional , Huesos/patología , Colágeno Tipo I/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Osteoblastos/patología , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/patología , Procolágeno/genética , Biosíntesis de Proteínas , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Transporte de Proteínas , Índice de Severidad de la EnfermedadRESUMEN
Hepatic stellate cells (HSCs) are thought to play key roles in the development of liver fibrosis. Extensive evidence has established the concept that αV integrins are involved in the activation of latent transforming growth factor ß (TGF-ß), a master regulator of the fibrotic signaling cascade. Based on mRNA and protein expression profiling data, we found that αVß1 integrin is the most abundant member of the αV integrin family in either quiescent or TGF-ß1-activated primary human HSCs. Unexpectedly, either a selective αVß1 inhibitor, Compound 8 (C8), or a pan-αV integrin inhibitor, GSK3008348, decreased TGF-ß1-activated procollagen I production in primary human HSCs, in which the role of ß1 integrin was confirmed by ITGB1 siRNA. In contrast with an Activin receptor-like kinase 5 (Alk5) inhibitor, C8 and GSK3008348 failed to inhibit TGF-ß1 induced SMAD3 and SMAD2 phosphorylation, but inhibited TGF-ß-induced phosphorylation of ERK1/2 and STAT3, suggesting that αVß1 integrin is involved in non-canonical TGF-ß signaling pathways. Consistently, ITGB1 siRNA significantly decreased phosphorylation of ERK1/2. Furthermore, a selective inhibitor of MEK1/2 blocked TGF-ß1 induced phosphorylation of ERK1/2 and decreased TGF-ß1 induced procollagen I production, while a specific inhibitor of STAT3 had no effect on TGF-ß1 induced procollagen I production. Taken together, current data indicate that αVß1 integrin can regulate TGF-ß signaling independent of its reported role in activating latent TGF-ß. Our data further support that αVß1 inhibition is a promising therapeutic target for the treatment of liver fibrosis.
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Células Estrelladas Hepáticas/metabolismo , Integrina alfa5beta1/genética , Procolágeno/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Proteína Smad2/genética , Factor de Crecimiento Transformador beta1/genética , Butiratos/farmacología , Regulación de la Expresión Génica , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , Humanos , Integrina alfa5beta1/antagonistas & inhibidores , Integrina alfa5beta1/metabolismo , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/genética , MAP Quinasa Quinasa 2/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Naftiridinas/farmacología , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Procolágeno/metabolismo , Pirazoles/farmacología , Pirrolidinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) has a collagen-rich dense extracellular matrix (ECM) that promotes malignancy of cancer cells and presents a barrier for drug delivery. Data analysis of our published mass spectrometry (MS)-based studies on enriched ECM from samples of progressive PDAC stages reveal that the C-terminal prodomains of fibrillar collagens are partially uncleaved in PDAC ECM, suggesting reduced procollagen C-proteinase activity. We further show that the enzyme responsible for procollagen C-proteinase activity, bone morphogenetic protein1 (BMP1), selectively suppresses tumor growth and metastasis in cells expressing high levels of COL1A1. Although BMP1, as a secreted proteinase, promotes fibrillar collagen deposition from both cancer cells and stromal cells, only cancer-cell-derived procollagen cleavage and deposition suppresses tumor malignancy. These studies reveal a role for cancer-cell-derived fibrillar collagen in selectively restraining tumor growth and suggest stratification of patients based on their tumor epithelial collagen I expression when considering treatments related to perturbation of fibrillar collagens.
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Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Colágenos Fibrilares/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Animales , Proteína Morfogenética Ósea 1/metabolismo , Carcinoma Ductal Pancreático/secundario , Línea Celular Tumoral , Colágeno Tipo I/química , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Progresión de la Enfermedad , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Colágenos Fibrilares/química , Colágenos Fibrilares/genética , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Mutagénesis , Neoplasias Pancreáticas/genética , Procolágeno/química , Procolágeno/genética , Procolágeno/metabolismo , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Dominant and recessive autosomal pathogenic variants in the three major genes (COL6A1-A2-A3) encoding the extracellular matrix protein collagen VI underlie a group of myopathies ranging from early-onset severe conditions (Ullrich congenital muscular dystrophy) to milder forms maintaining independent ambulation (Bethlem myopathy). Diagnosis is based on the combination of clinical presentation, muscle MRI, muscle biopsy, analysis of collagen VI secretion, and COL6A1-A2-A3 genetic analysis, the interpretation of which can be challenging. OBJECTIVE: To refine the phenotypical spectrum associated with the frequent COL6A3 missense variant c.7447A>G (p.Lys2483Glu). METHODS: We report the clinical and molecular findings in 16 patients: 12 patients carrying this variant in compound heterozygosity with another COL6A3 variant, and four homozygous patients. RESULTS: Patients carrying this variant in compound heterozygosity with a truncating COL6A3 variant exhibit a phenotype consistent with COL6-related myopathies (COL6-RM), with joint contractures, proximal weakness and skin abnormalities. All remain ambulant in adulthood and only three have mild respiratory involvement. Most show typical muscle MRI findings. In five patients, reduced collagen VI secretion was observed in skin fibroblasts cultures. All tested parents were unaffected heterozygous carriers. Conversely, two out of four homozygous patients did not present with the classical COL6-RM clinical and imaging findings. Collagen VI immunolabelling on cultured fibroblasts revealed rather normal secretion in one and reduced secretion in another. Muscle biopsy from one homozygous patient showed myofibrillar disorganization and rimmed vacuoles. CONCLUSIONS: In light of our results, we postulate that the COL6A3 variant c.7447A>G may act as a modulator of the clinical phenotype. Thus, in patients with a typical COL6-RM phenotype, a second variant must be thoroughly searched for, while for patients with atypical phenotypes further investigations should be conducted to exclude alternative causes. This works expands the clinical and molecular spectrum of COLVI-related myopathies.
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Colágeno Tipo VI/genética , Distrofias Musculares/genética , Procolágeno/genética , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Heterocigoto , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Músculo Esquelético/patología , Enfermedades Musculares/genética , Mutación , Fenotipo , Adulto JovenRESUMEN
Intracellular procollagen folding begins at the protein's C-terminal propeptide (C-Pro) domain, which initiates triple-helix assembly and defines the composition and chain register of fibrillar collagen trimers. The C-Pro domain is later proteolytically cleaved and excreted from the body, while the mature triple helix is incorporated into the extracellular matrix. The procollagen C-Pro domain possesses a single N-glycosylation site that is widely conserved in all the fibrillar procollagens across humans and diverse other species. Given that the C-Pro domain is removed once procollagen folding is complete, the N-glycan might be presumed to be important for folding. Surprisingly, however, there is no difference in the folding and secretion of N-glycosylated versus non-N-glycosylated collagen type-I, leaving the function of the N-glycan unclear. We hypothesized that the collagen N-glycan might have a context-dependent function, specifically, that it could be required to promote procollagen folding only when proteostasis is challenged. We show that removal of the N-glycan from misfolding-prone C-Pro domain variants does indeed cause serious procollagen and ER proteostasis defects. The N-glycan promotes folding and secretion of destabilized C-Pro variants by providing access to the ER's lectin-based chaperone machinery. Finally, we show that the C-Pro N-glycan is actually critical for the folding and secretion of even wild-type procollagen under ER stress conditions. Such stress is commonly incurred during development, wound healing, and other processes in which collagen production plays a key role. Collectively, these results establish an essential, context-dependent function for procollagen's previously enigmatic N-glycan, wherein the carbohydrate moiety buffers procollagen folding against proteostatic challenge.
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Matriz Extracelular/metabolismo , Procolágeno/metabolismo , Proteoglicanos/metabolismo , Proteostasis , Colágeno , Matriz Extracelular/genética , Glicosilación , Humanos , Procolágeno/genética , Dominios Proteicos , Proteoglicanos/genéticaRESUMEN
BACKGROUND: Sarcopenia is a multifactorial pathophysiologic condition of skeletal muscle mass and muscle strength associated with aging. However, biomarkers for predicting the occurrence of sarcopenia are rarely discussed in recent studies. The aim of the study was to elucidate the relationship between sarcopenia and several pertinent biomarkers. METHODS: Using the Gene Expression Omnibus (GEO) profiles of the National Center for Biotechnology Information, the associations between mRNA expression of biomarkers and sarcopenia were explored, including high temperature requirement serine protease A1 (HtrA1), procollagen type III N-terminal peptide (P3NP), apelin, and heat shock proteins 70 (Hsp72). We enrolled 408 community-dwelling adults aged 65 years and older with sarcopenia and nonsarcopenia based on the algorithm proposed by the Asian Working Group for Sarcopenia (AWGS). Muscle strength is identified by hand grip strength using an analogue isometric dynamometer. Muscle mass is estimated by skeletal mass index (SMI) using a bioelectrical impedance analysis. Physical performance is measured by gait speed using 6 m walking distance. The associations between these biomarkers and sarcopenia were determined using receiver operating characteristic (ROC) curve analysis and multivariate regression models. RESULTS: From the GEO profiles, the sarcopenia gene set variation analysis score was correlated significantly with the mRNA expression of APLNR (p < 0.001) and HSPA2 (p < 0.001). In our study, apelin was significantly associated with decreased hand grip strength with ß values of - 0.137 (95%CI: - 0.229, - 0.046) in men. P3NP and HtrA1 were significantly associated with increased SMI with ß values of 0.081 (95%CI: 0.010, 0.153) and 0.005 (95%CI: 0.001, 0.009) in men, respectively. Apelin and HtrA1 were inversely associated with the presence of sarcopenia with an OR of 0.543 (95%CI: 0.397-0.743) and 0.003 (95%CI: 0.001-0.890) after full adjustment. The cutoff point of HtrA1 was associated with the presence of sarcopenia with an OR of 0.254 (95%CI: 0.083-0.778) in men. The cutoff point of apelin was negatively associated with the presence of sarcopenia with an OR of 0.254 (95%CI: 0.083-0.778). CONCLUSION: Our study highlights that P3NP, HtrA, and apelin are useful for diagnosis of sarcopenia in the clinical setting.
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Apelina/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas/metabolismo , Fragmentos de Péptidos/metabolismo , Procolágeno/metabolismo , Sarcopenia , Anciano , Apelina/genética , Estudios Transversales , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Fuerza de la Mano , Serina Peptidasa A1 que Requiere Temperaturas Altas/genética , Humanos , Masculino , Fuerza Muscular , Músculo Esquelético , Fragmentos de Péptidos/genética , Procolágeno/genética , Sarcopenia/diagnóstico , Sarcopenia/epidemiología , Sarcopenia/genéticaRESUMEN
Proteolytic processing of procollagens is a central step during collagen fibril formation. Bone morphogenic protein 1 (BMP1) is a metalloprotease that plays an important role in the cleavage of carboxy-terminal (COOH-terminal) propeptides from procollagens. Although the removal of propeptides is required to generate mature collagen fibrils, the contribution of BMP1 to this proteolytic process and its action site remain to be fully determined. In this study, using postnatal lung fibroblasts as a model system, we showed that genetic ablation of Bmp1 in primary murine lung fibroblasts abrogated COOH-terminal cleavage from type I procollagen as measured by COOH-terminal propeptide of type I procollagen (CICP) production. We also showed that inhibition of BMP1 by siRNA-mediated knockdown or small-molecule inhibitor reduced the vast majority of CICP production and collagen deposition in primary human lung fibroblasts. Furthermore, we discovered and characterized two antibody inhibitors for BMP1. In both postnatal lung fibroblast and organoid cultures, BMP1 blockade prevented CICP production. Together, these findings reveal a nonredundant role of extracellular BMP1 to process CICP in lung fibroblasts and suggest that development of antibody inhibitors is a viable pharmacological approach to target BMP1 proteinase activity in fibrotic diseases.
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Proteína Morfogenética Ósea 1/metabolismo , Líquido Extracelular/metabolismo , Fibroblastos/metabolismo , Pulmón/metabolismo , Fragmentos de Péptidos/metabolismo , Procolágeno/metabolismo , Proteolisis , Secuencia de Aminoácidos , Animales , Proteína Morfogenética Ósea 1/antagonistas & inhibidores , Proteína Morfogenética Ósea 1/genética , Células CHO , Cricetinae , Cricetulus , Líquido Extracelular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Células HEK293 , Humanos , Pulmón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Organoides , Oxadiazoles/farmacología , Fragmentos de Péptidos/genética , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Procolágeno/genética , Inhibidores de Proteasas/farmacología , Proteolisis/efectos de los fármacos , ConejosRESUMEN
The effect of plyometric exercise on bone biomarkers has been studied in pediatric and young adult populations in order to better understand how exercise influences bone homeostasis. However, there are no such data in postmenopausal women, a group characterized by an uncoupling of the bone resorption-formation cycle. This study examined the serum concentrations of sclerostin, dickkopf-1 (DKK1), c-terminal crosslinking telopeptides of type I collagen (CTXI), and procollagen type I amino-terminal propeptide (PINP) at rest and following a single bout of plyometric exercise in 20 premenopausal (23.1 ± 2.3 years) and 20 postmenopausal women (57.9 ± 4.3 years). The exercise consisted of 128 jumps, organized into 5 circuit stations. Blood samples were obtained prior to and 5 min, 1 h, and 24 h postexercise. At rest, postmenopausal women had significantly higher sclerostin and CTXI, but lower DKK1 than premenopausal women. Sclerostin increased 5 min postexercise only in the premenopausal group. DKK1 decreased 24 h postexercise in the premenopausal women while it decreased 1 h postexercise in the postmenopausal women. In both groups, CTXI did not change across time and PINP decreased 5 min and 1 h postexercise (p < 0.05). The PINP/CTXI ratio decreased 5 min and 1 h postexercise then significantly increased 24 h postexercise only in premenopausal women. These results indicate that although plyometric exercise is effective in eliciting osteoanabolic effects in younger women; such an effect is not evident in postmenopausal women.
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Proteínas Adaptadoras Transductoras de Señales/genética , Colágeno Tipo I/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Fragmentos de Péptidos/genética , Péptidos/genética , Posmenopausia/sangre , Premenopausia/sangre , Procolágeno/genética , Proteínas Adaptadoras Transductoras de Señales/sangre , Tejido Adiposo/fisiología , Anciano , Biomarcadores/sangre , Índice de Masa Corporal , Resorción Ósea/genética , Resorción Ósea/metabolismo , Colágeno Tipo I/sangre , Estradiol/sangre , Femenino , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/sangre , Persona de Mediana Edad , Osteogénesis/genética , Fragmentos de Péptidos/sangre , Péptidos/sangre , Ejercicio Pliométrico/métodos , Procolágeno/sangreRESUMEN
UVB irradiation induces pro-inflammatory cytokines including interleukin-1 (IL-1) and tumor necrosis factor-α (TNFα) in the skin. TNFα stimulates the chemotaxis of inflammatory cells to the skin. These cells secrete metalloproteinases (MMPs) and other enzymes that damage the cutaneous matrix. Therefore, blocking TNFα activity could be effective in preventing the influx of inflammatory cells and subsequent collagen degradation in the skin. In addition, TNFα downregulates procollagen mRNA, and thus blockade may be beneficial to production of type I collagen. Female C57BL/6 J mice were treated with etanercept (TNFα blocker, 4 mg/kg/day) for 4 days 1 h prior to UVB irradiation (100 mJ/cm2/day for 5 days). On the 5th day mice were sacrificed 3 h after UVB exposure. Blocking TNFα significantly inhibited UVB-induced recruitment of macrophages, mast cells, and neutrophils. UVB-irradiated mice skin contained more mature collagen compared to etanercept and UVB + etanercept-treated mice. Skin from UVB + etanercept-treated mice had more collagen fragments relative to UVB-irradiated mice. Procollagen protein was lower in UVB-irradiated and UVB + etanercept-treated mice. TNFα blockade decreased decorin and TGF-ß1 in UVB-irradiated mice compared to UVB alone. MMP13 was inhibited by etanercept in UVB-irradiated mice (p < 0.01). In conclusion, blockade of TNFα significantly decreased mature collagen in UVB-irradiated mice, while increasing collagen fragmentation and decreasing procollagen.
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Colágeno Tipo I/metabolismo , Factor de Necrosis Tumoral alfa/efectos de la radiación , Animales , Movimiento Celular , Decorina/metabolismo , Femenino , Expresión Génica , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Procolágeno/genética , Proteolisis , ARN Mensajero , Piel , Envejecimiento de la Piel , Factor de Crecimiento Transformador beta1/metabolismo , Rayos UltravioletaRESUMEN
The Ehlers-Danlos syndromes (EDS) are a heterogeneous group of hereditary disorders of connective tissue, with common features including joint hypermobility, soft and hyperextensible skin, abnormal wound healing and easy bruising. Fourteen different types of EDS are recognized, of which the molecular cause is known for 13 types. These types are caused by variants in 20 different genes, the majority of which encode the fibrillar collagen types I, III and V, modifying or processing enzymes for those proteins, and enzymes that can modify glycosaminoglycan chains of proteoglycans. For the hypermobile type of EDS, the molecular underpinnings remain unknown. As connective tissue is ubiquitously distributed throughout the body, manifestations of the different types of EDS are present, to varying degrees, in virtually every organ system. This can make these disorders particularly challenging to diagnose and manage. Management consists of a care team responsible for surveillance of major and organ-specific complications (for example, arterial aneurysm and dissection), integrated physical medicine and rehabilitation. No specific medical or genetic therapies are available for any type of EDS.