Col6a1 null mice as a model to study skin phenotypes in patients with collagen VI related myopathies: expression of classical and novel collagen VI variants during wound healing.
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ABSTRACT:
SUBMITTER: Lettmann S
PROVIDER: S-EPMC4144880 | biostudies-literature | 2014
REPOSITORIES: biostudies-literature
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Project description:Cutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-? (TGF-?) signaling, and mice lacking components of the TGF-? signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-? pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4(NULL) mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4(NULL) mice, resolution of inflammation was delayed, and expression of ?4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4(NULL) skin. The induced level of phosphorylated Smad2 in response to TGF-? was reduced in cultured CLIC4(NULL) keratinocytes relative to in wild-type cells, and CLIC4(NULL) keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-?. CLIC4(NULL) keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-? pathway.
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Project description:Carbonic anhydrases (CAs) contribute to tumor cell migration by generating an acidic environment through the conversion of carbon dioxide to bicarbonate and a proton. CA VI is secreted to milk and saliva, and it could contribute to wound closure, as a potential trophic factor, in animals that typically lick their wounds. Our aim was to investigate whether human CA VI improves skin-wound healing in full-thickness skin-wound models. The effect was studied in Car6 -/- knockout mice and wild type littermates. Half of both mice strains were given topically administered, milk-derived CA VI after wounding and eight hours later. The amount of topically given CA VI exceeded the predicted amount of natural saliva-delivered CA VI. The healing was followed for seven days and studied from photographs and histological sections. Our results showed no significant differences between the treatment groups in wound closure, re-epithelization, or granulation tissue formation, nor did the Car6 genotype affect the healing. Our results demonstrate that CA VI does not play a major role in skin-wound healing and also suggest that saliva-derived CA VI is not responsible for the licking-associated improved wound healing in animals.
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Project description:Collagen VI is an integral part of the skeletal muscle extracellular matrix, providing mechanical stability and facilitating matrix-dependent cell signaling. Mutations in collagen VI result in either Ullrich congenital muscular dystrophy (UCMD) or Bethlem myopathy (BM), with UCMD being clinically more severe. Recent studies demonstrating increased apoptosis and abnormal mitochondrial function in Col6a1 knockout mice and in human myoblasts have provided the first mechanistic insights into the pathophysiology of these diseases. However, how loss of collagen VI causes mitochondrial dysfunction remains to be understood. Progress is hindered in part by the lack of an adequate animal model for UCMD, as knockout mice have a mild motor phenotype. To further the understanding of these disorders, we have generated zebrafish models of the collagen VI myopathies. Morpholinos designed to exon 9 of col6a1 produced a severe muscle disease reminiscent of UCMD, while ones to exon 13 produced a milder phenotype similar to BM. UCMD-like zebrafish have increased cell death and abnormal mitochondria, which can be attenuated by treatment with the proton pump modifier cyclosporin A (CsA). CsA improved the motor deficits in UCMD-like zebrafish, but failed to reverse the sarcolemmal membrane damage. In all, we have successfully generated the first vertebrate model matching the clinical severity of UCMD and demonstrated that CsA provides phenotypic improvement, thus corroborating data from knockout mice supporting the use of mitochondrial permeability transition pore modifiers as therapeutics in patients, and providing proof of principle for the utility of the zebrafish as a powerful preclinical model.
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Project description:Glycine substitutions in the conserved Gly-X-Y motif in the triple helical (TH) domain of collagen VI are the most commonly identified mutations in the collagen VI myopathies including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate (INT) phenotypes. We describe clinical and genetic characteristics of 97 individuals with glycine substitutions in the TH domain of COL6A1, COL6A2, or COL6A3 and add a review of 97 published cases, for a total of 194 cases. Clinical findings include severe, INT, and mild phenotypes even from patients with identical mutations. INT phenotypes were most common, accounting for almost half of patients, emphasizing the importance of INT phenotypes to the overall phenotypic spectrum. Glycine substitutions in the TH domain are heavily clustered in a short segment N-terminal to the 17th Gly-X-Y triplet, where they are acting as dominants. The most severe cases are clustered in an even smaller region including Gly-X-Y triplets 10-15, accounting for only 5% of the TH domain. Our findings suggest that clustering of glycine substitutions in the N-terminal region of collagen VI is not based on features of the primary sequence. We hypothesize that this region may represent a functional domain within the triple helix.
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Project description:Collagen VI is a non-fibrillar collagen present in the extracellular matrix (ECM) as a complex polymer; the mainly expressed form is composed of α1, α2 and α3 chains; mutations in genes encoding these chains cause myopathies known as Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and myosclerosis myopathy (MM). The collagen VI α6 chain is a recently identified component of the ECM of the human skeletal muscle. Here we report that the α6 chain was dramatically reduced in skeletal muscle and muscle cell cultures of genetically characterized UCMD, BM and MM patients, independently of the clinical phenotype, the gene involved and the effect of the mutation on the expression of the "classical" α1α2α3 heterotrimer. By contrast, the collagen VI α6 chain was normally expressed or increased in the muscle of patients affected by other forms of muscular dystrophy, the overexpression matching with areas of increased fibrosis. In vitro treatment with TGF-β1, a potent collagen inducer, promoted the collagen VI α6 chain deposition in the ECM of normal muscle cells, whereas, in cultures derived from collagen VI-related myopathy patients, the collagen VI α6 chain failed to develop a network outside the cells and accumulated in the endoplasmic reticulum. The defect of the α6 chain points to a contribution to the pathogenesis of collagen VI-related disorders.
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Project description:MED1 (Mediator complex subunit 1) is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1(epi-/-)) that develop epidermal hyperplasia. Herein, to investigate the function(s) of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1(epi-/-) and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1(epi-/-) mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1(epi-/-) mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1(epi-/-) keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1(epi-/-) keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1(epi-/-) keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1(epi-/-) keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1(epi-/-) mice. On the other hand, skin wound healing in 6-month-old Med1(epi-/-) mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1(epi-/-) mice, indicating a decreased contribution of hair follicle stem cells to epidermal regeneration after wounding in 6-month-old Med1(epi-/-) mice. This study sheds light on the novel function of MED1 in keratinocytes and suggests a possible new therapeutic approach for skin wound healing and aging.
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Project description:The spectrum of clinical phenotypes associated with a deficiency or dysfunction of collagen VI in the extracellular matrix of muscle are collectively termed 'collagen VI-related myopathies' and include Ullrich congenital muscular dystrophy, Bethlem myopathy and intermediate phenotypes. To further define the clinical course of these variants, we studied the natural history of pulmonary function in correlation to motor abilities in the collagen VI-related myopathies by analysing longitudinal forced vital capacity data in a large international cohort. Retrospective chart reviews of genetically and/or pathologically confirmed collagen VI-related myopathy patients were performed at 10 neuromuscular centres: USA (n = 2), UK (n = 2), Australia (n = 2), Italy (n = 2), France (n = 1) and Belgium (n = 1). A total of 486 forced vital capacity measurements obtained in 145 patients were available for analysis. Patients at the severe end of the clinical spectrum, conforming to the original description of Ullrich congenital muscular dystrophy were easily identified by severe muscle weakness either preventing ambulation or resulting in an early loss of ambulation, and demonstrated a cumulative decline in forced vital capacity of 2.6% per year (P < 0.0001). Patients with better functional abilities, in whom walking with/without assistance was achieved, were initially combined, containing both intermediate and Bethlem myopathy phenotypes in one group. However, one subset of patients demonstrated a continuous decline in pulmonary function whereas the other had stable pulmonary function. None of the patients with declining pulmonary function attained the ability to hop or run; these patients were categorized as intermediate collagen VI-related myopathy and the remaining patients as Bethlem myopathy. Intermediate patients had a cumulative decline in forced vital capacity of 2.3% per year (P < 0.0001) whereas the relationship between age and forced vital capacity in patients with Bethlem myopathy was not significant (P = 0.1432). Nocturnal non-invasive ventilation was initiated in patients with Ullrich congenital muscular dystrophy by 11.3 years (±4.0) and in patients with intermediate collagen VI-related myopathy by 20.7 years (±1.5). The relationship between maximal motor ability and forced vital capacity was highly significant (P < 0.0001). This study demonstrates that pulmonary function profiles can be used in combination with motor function profiles to stratify collagen VI-related myopathy patients phenotypically. These findings improve our knowledge of the natural history of the collagen VI-related myopathies, enabling proactive optimization of care and preparing this patient population for clinical trials.
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