Creation and characterization of novel rat model for recessive dystrophic epidermolysis bullosa: Frameshift mutation of the Col7a1 gene leads to severe blistered phenotype.

Recessive dystrophic epidermolysis bullosa is a rare genodermatosis caused by a mutation of the Col7a1 gene. The Col7a1 gene codes for collagen type VII protein, a major component of anchoring fibrils. Mutations of the Col7a1 gene can cause aberrant collagen type VII formation, causing an associated lack or absence of anchoring fibrils. This presents clinically as chronic blistering, scarring, and fibrosis, often leading to the development of cutaneous squamous cell carcinoma. Patients also experience persistent pain and pruritus. Pain management and supportive bandaging remain the primary treatment options. The pathology of recessive dystrophic epidermolysis bullosa was first described in the 1980s, and there has since been a multitude of encouraging treatment options developed. However, in vivo research has been hindered by inadequate models of the disease. The various mouse models in existence possess longevity and surface area constraints, or do not adequately model a normal human disease state. In this paper, we describe a novel rat model of recessive dystrophic epidermolysis bullosa that offers an alternative to previous murine models. An 8-base pair deletion was induced in the Col7a1 gene of Lewis rats, which was subsequently found to cause a premature stop codon downstream. Homozygous mutants presented with a fragile and chronically blistered phenotype postnatally. Further histological analysis revealed subepidermal clefting and the absence of anchoring fibrils. The generation of this novel model offers researchers an easily maintained organism that possesses a larger surface area for experimental topical and transfused therapies to be tested, which may provide great utility in the future study of this debilitating disease.

Characterization of TGFß1-induced tendon-like structure in the scaffold-free three-dimensional tendon cell culture system.

The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells in a differentially adherent growth channel. Transforming Growth Factor-ß (TGFß) signaling is involved in various biological processes in the tendon, regulating tendon cell fate, recruitment and maintenance of tenocytes, and matrix organization. This known function of TGFß signaling in tendon prompted us to utilize TGFß1 to induce tendon-like structures in 3D tendon constructs. TGFß1 treatment promoted a tendon-like structure in the peripheral layer of the constructs characterized by increased thickness with a gradual decrease in cell density and highly aligned collagen matrix. TGFß1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFß1 treatment also induced early tenogenic differentiation and resulted in sufficient mechanical integrity, allowing biomechanical testing. The current study suggests that this scaffold-free 3D tendon cell culture system could be an in vitro platform to investigate underlying biological mechanisms that regulate tenogenic cell differentiation and matrix organization.

Unraveling the role of TGFß signaling in thoracic aortic aneurysm and dissection using Fbn1 mutant mouse models.

Although abnormal TGFß signaling is observed in several heritable forms of thoracic aortic aneurysms and dissections including Marfan syndrome, its precise role in aortic disease progression is still disputed. Using a mouse genetic approach and quantitative isobaric labeling proteomics, we sought to elucidate the role of TGFß signaling in three Fbn1 mutant mouse models representing a range of aortic disease from microdissection (without aneurysm) to aneurysm (without rupture) to aneurysm and rupture. Results indicated that reduced TGFß signaling and increased mast cell proteases were associated with microdissection. In contrast, increased abundance of extracellular matrix proteins, which could be reporters for positive TGFß signaling, were associated with aneurysm. Marked reductions in collagens and fibrillins, and increased TGFß signaling, were associated with aortic rupture. Our data indicate that TGFß signaling performs context-dependent roles in the pathogenesis of thoracic aortic disease.

The Fraser Complex Proteins (Frem1, Frem2, and Fras1) Can Form Anchoring Cords in the Absence of AMACO at the Dermal-Epidermal Junction of Mouse Skin.

AMACO (VWA2 protein), secreted by epithelial cells, is strongly expressed at basement membranes when budding or invagination occurs in embryos. In skin, AMACO associates with proteins of the Fraser complex, which form anchoring cords. These, during development, temporally stabilize the dermal-epidermal junction, pending the formation of collagen VII-containing anchoring fibrils. Fraser syndrome in humans results if any of the core members of the Fraser complex (Fras1, Frem1, Frem2) are mutated. Fraser syndrome is characterized by subepidermal blistering, cryptophthalmos, and syndactyly. In an attempt to determine AMACO function, we generated and characterized AMACO-deficient mice. In contrast to Fraser complex mutant mice, AMACO-deficient animals lack an obvious phenotype. The mutually interdependent basement membrane deposition of the Fraser complex proteins, and the formation of anchoring cords, are not affected. Furthermore, hair follicle development in newborn AMACO-deficient mice showed no gross aberration. Surprisingly, it appears that, while AMACO is a component of the anchoring cords, it is not essential for their formation or function.

Lysyl hydroxylase 3-mediated post-translational modifications are required for proper biosynthesis of collagen α1α1α2(IV).

Collagens are the most abundant proteins in the body and among the most biosynthetically complex. A molecular ensemble of over 20 endoplasmic reticulum resident proteins participates in collagen biosynthesis and contributes to heterogeneous post-translational modifications. Pathogenic variants in genes encoding collagens cause connective tissue disorders, including osteogenesis imperfecta, Ehlers-Danlos syndrome, and Gould syndrome (caused by mutations in COL4A1 and COL4A2), and pathogenic variants in genes encoding proteins required for collagen biosynthesis can cause similar but overlapping clinical phenotypes. Notably, pathogenic variants in lysyl hydroxylase 3 (LH3) cause a multisystem connective tissue disorder that exhibits pathophysiological features of collagen-related disorders. LH3 is a multifunctional collagen-modifying enzyme; however, its precise role(s) and substrate specificity during collagen biosynthesis has not been defined. To address this critical gap in knowledge, we generated LH3 KO cells and performed detailed quantitative and molecular analyses of collagen substrates. We found that LH3 deficiency severely impaired secretion of collagen α1α1α2(IV) but not collagens α1α1α2(I) or α1α1α1(III). Amino acid analysis revealed that LH3 is a selective LH for collagen α1α1α2(IV) but a general glucosyltransferase for collagens α1α1α2(IV), α1α1α2(I), and α1α1α1(III). Importantly, we identified rare variants that are predicted to be pathogenic in the gene encoding LH3 in two of 113 fetuses with intracranial hemorrhage-a cardinal feature of Gould syndrome. Collectively, our findings highlight a critical role of LH3 in α1α1α2(IV) biosynthesis and suggest that LH3 pathogenic variants might contribute to Gould syndrome.

EMILIN1 deficiency causes arterial tortuosity with osteopenia and connects impaired elastogenesis with defective collagen fibrillogenesis.

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.

Vertebrate extracellular matrix protein hemicentin-1 interacts physically and genetically with basement membrane protein nidogen-2.

Hemicentins are large proteins of the extracellular matrix that belong to the fibulin family and play pivotal roles during development and homeostasis of a variety of invertebrate and vertebrate tissues. However, bona fide interaction partners of hemicentins have not been described as yet. Here, applying surface plasmon resonance spectroscopy and co-immunoprecipitation, we identify the basement membrane protein nidogen-2 (NID2) as a binding partner of mouse and zebrafish hemicentin-1 (HMCN1), in line with the formerly described essential role of mouse HMCN1 in basement membrane integrity. We show that HMCN1 binds to the same protein domain of NID2 (G2) as formerly shown for laminins, but with an approximately 3.5-fold lower affinity and in a competitive manner. Furthermore, immunofluorescence and immunogold labeling revealed that HMCN1/Hmcn1 is localized close to basement membranes and in partial overlap with NID2/Nid2a in different tissues of mouse and zebrafish. Genetic knockout and antisense-mediated knockdown studies in zebrafish further show that loss of Nid2a leads to similar defects in fin fold morphogenesis as the loss of Laminin-α5 (Lama5) or Hmcn1. Finally, combined partial loss-of-function studies indicated that nid2a genetically interacts with both hmcn1 and lama5. Together, these findings suggest that despite their mutually exclusive physical binding, hemicentins, nidogens, and laminins tightly cooperate and support each other during formation, maintenance, and function of basement membranes to confer tissue linkage.

Anchoring Cords: A Distinct Suprastructure in the Developing Skin.

AMACO (VWA2 protein) is a basement membrane-associated protein secreted by epithelial cells. It is strongly expressed when invagination or budding occurs during development. AMACO associates with the Fraser complex, which when mutated causes Fraser syndrome, characterized by subepidermal blistering, cryptophthalmos, and syndactyly. The core Fraser complex proteins FRAS1, FREM1, and FREM2 localize at the dermal‒epidermal junction and mediate adhesion to the underlying dermis during embryonic development. Earlier transmission electron microscopy studies of adult mouse skin showed clustered AMACO deposition below the lamina densa. In this study, we report a distinct cord-like suprastructure in the neonate dermis to which AMACO- and Fraser complex‒associated proteins contribute. We propose anchoring cords to designate the suprastructure. Anchoring cords have a diameter of 60 nm when immunolabeled, originate from the basement membrane, and extend several microns into the dermis. In normal skin, they are evident after immunogold electron microscopy and are strikingly appreciated in thicker sections. In recessive dystrophic epidermolysis bullosa skin, they are directly visible where collagen VII anchoring fibrils are ablated. Immunofluorescence and coimmunoprecipitation of skin extracts identify a direct interaction of FREM2 and AMACO.

Elevated TGFß signaling contributes to ocular anterior segment dysgenesis in Col4a1 mutant mice.

Ocular anterior segment dysgenesis (ASD) refers to a collection of developmental disorders affecting the anterior structures of the eye. Although a number of genes have been implicated in the etiology of ASD, the underlying pathogenetic mechanisms remain unclear. Mutations in genes encoding collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) cause Gould syndrome, a multi-system disorder that often includes ocular manifestations such as ASD and glaucoma. COL4A1 and COL4A2 are abundant basement membrane proteins that provide structural support to tissues and modulate signaling through interactions with other extracellular matrix proteins, growth factors, and cell surface receptors. In this study, we used a combination of histological, molecular, genetic and pharmacological approaches to demonstrate that altered TGFß signaling contributes to ASD in mouse models of Gould syndrome. We show that TGFß signaling was elevated in anterior segments from Col4a1 mutant mice and that genetically reducing TGFß signaling partially prevented ASD. Notably, we identified distinct roles for TGFß1 and TGFß2 in ocular defects observed in Col4a1 mutant mice. Importantly, we show that pharmacologically promoting type IV collagen secretion or reducing TGFß signaling ameliorated ocular pathology in Col4a1 mutant mice. Overall, our findings demonstrate that altered TGFß signaling contributes to COL4A1-related ocular dysgenesis and implicate this pathway as a potential therapeutic target for the treatment of Gould syndrome.

In vivo topical gene therapy for recessive dystrophic epidermolysis bullosa: a phase 1 and 2 trial.

Recessive dystrophic epidermolysis bullosa (RDEB) is a lifelong genodermatosis associated with blistering, wounding, and scarring caused by mutations in COL7A1, the gene encoding the anchoring fibril component, collagen VII (C7). Here, we evaluated beremagene geperpavec (B-VEC), an engineered, non-replicating COL7A1 containing herpes simplex virus type 1 (HSV-1) vector, to treat RDEB skin. B-VEC restored C7 expression in RDEB keratinocytes, fibroblasts, RDEB mice and human RDEB xenografts. Subsequently, a randomized, placebo-controlled, phase 1 and 2 clinical trial (NCT03536143) evaluated matched wounds from nine RDEB patients receiving topical B-VEC or placebo repeatedly over 12 weeks. No grade 2 or above B-VEC-related adverse events or vector shedding or tissue-bound skin immunoreactants were noted. HSV-1 and C7 antibodies sometimes presented at baseline or increased after B-VEC treatment without an apparent impact on safety or efficacy. Primary and secondary objectives of C7 expression, anchoring fibril assembly, wound surface area reduction, duration of wound closure, and time to wound closure following B-VEC treatment were met. A patient-reported pain-severity secondary outcome was not assessed given the small proportion of wounds treated. A global assessment secondary endpoint was not pursued due to redundancy with regard to other endpoints. These studies show that B-VEC is an easily administered, safely tolerated, topical molecular corrective therapy promoting wound healing in patients with RDEB.

A new murine model of Barth syndrome neutropenia links TAFAZZIN deficiency to increased ER stress-induced apoptosis.

Barth syndrome is an inherited X-linked disorder that leads to cardiomyopathy, skeletal myopathy, and neutropenia. These symptoms result from the loss of function of the enzyme TAFAZZIN, a transacylase located in the inner mitochondrial membrane that is responsible for the final steps of cardiolipin production. The link between defective cardiolipin maturation and neutropenia remains unclear. To address potential mechanisms of neutropenia, we examined myeloid progenitor development within the fetal liver of TAFAZZIN knockout (KO) animals as well as within the adult bone marrow of wild-type recipients transplanted with TAFAZZIN-KO hematopoietic stem cells. We also used the ER-Hoxb8 system (estrogen receptor fused to Hoxb8) of conditional immortalization to establish a new murine model system for the ex vivo study of TAFAZZIN-deficient neutrophils. The TAFAZZIN-KO cells demonstrated the expected dramatic differences in cardiolipin maturation that result from a lack of TAFAZZIN enzyme activity. Contrary to our hypothesis, we did not identify any significant differences in neutrophil development or neutrophil function across a variety of assays including phagocytosis and the production of cytokines or reactive oxygen species. However, transcriptomic analysis of the TAFAZZIN-deficient neutrophil progenitors demonstrated an upregulation of markers of endoplasmic reticulum stress and confirmatory testing demonstrated that the TAFAZZIN-deficient cells had increased sensitivity to certain ER stress-mediated and non-ER stress-mediated triggers of apoptosis. Although the link between increased sensitivity to apoptosis and the variably penetrant neutropenia phenotype seen in some patients with Barth syndrome remains to be clarified, our studies and new model system set a foundation for further investigation.

Molecular alterations due to Col5a1 haploinsufficiency in a mouse model of classic Ehlers-Danlos syndrome.

Type V collagen is a regulatory fibrillar collagen essential for type I collagen fibril nucleation and organization and its deficiency leads to structurally abnormal extracellular matrix (ECM). Haploinsufficiency of the Col5a1 gene encoding α(1) chain of type V collagen is the primary cause of classic Ehlers-Danlos syndrome (EDS). The mechanisms by which this initial insult leads to the spectrum of clinical presentation are not fully understood. Using transcriptome analysis of skin and Achilles tendons from Col5a1 haploinsufficient (Col5a1+/-) mice, we recognized molecular alterations associated with the tissue phenotypes. We identified dysregulation of ECM components including thrombospondin-1, lysyl oxidase, and lumican in the skin of Col5a1+/- mice when compared with control. We also identified upregulation of transforming growth factor ß1 (Tgf-ß) in serum and increased expression of pSmad2 in skin from Col5a1+/- mice, suggesting Tgf-ß dysregulation is a contributor to abnormal wound healing and atrophic scarring seen in classic EDS. Together, these findings support altered matrix to cell signaling as a component of the pathogenesis of the tissue phenotype in classic EDS and point out potential downstream signaling pathways that may be targeted for the treatment of this disease.

Identification of Missense Extracellular Matrix Gene Variants in a Large Glaucoma Pedigree and Investigation of the N700S Thrombospondin-1 Variant in Normal and Glaucomatous Trabecular Meshwork Cells.

PURPOSE: Primary open-angle glaucoma (POAG) is a complex heterogeneous disease. While several POAG genes have been identified, a high proportion of estimated heritability remains unexplained. Elevated intraocular pressure (IOP) is a leading POAG risk factor and dysfunctional extracellular matrix (ECM) in the trabecular meshwork (TM) contributes to elevated IOP. In this study, we sought to identify missense variants in ECM genes that correlate with ocular hypertensive POAG. METHODS: Whole-genome sequencing was used to identify genetic variants in five members of a large POAG family (n = 68) with elevated IOP. The remaining family members were screened by Sanger sequencing. Unrelated normal (NTM) and glaucomatous (GTM) cells were sequenced for the identified variants. The ECM protein levels were determined by Western immunoblotting and confocal and electron microscopy investigated ECM ultrastructural organization. RESULTS: Three ECM gene variants were significantly associated with POAG or elevated IOP in a large POAG pedigree. These included rs2228262 (N700S; thrombospondin-1 (THBS1, TSP1)), rs112913396 (D563 G; collagen type VI, alpha 3 (COL6A3)) and rs34759087 (E987K; laminin subunit beta 2 (LAMB2)). Screening of unrelated TM cells (n = 27) showed higher prevalence of the THBS1 variant but not the LAMB2 variant, in GTM cells (39%) than NTM cells (11%). The rare COL6A3 variant was not detected. TSP1 protein was upregulated and COL6A3 was down-regulated in TM cells with N700S subject to mechanical stretch, an in vitro method that mimics elevated IOP. Immunofluorescence showed increased TSP1 immunostaining in cell strains with N700S compared to wild-type TM cells. Ultrastructural studies showed ECM disorganization and altered collagen type VI distribution in GTM versus NTM cells. CONCLUSIONS: Our results suggest that missense variants in ECM genes may not cause catastrophic changes to the TM, but over many years, subtle changes in ECM may accumulate and cause structural disorganization of the outflow resistance leading to elevated IOP in POAG patients.

A Novel Model of Tobacco Smoke-Mediated Aortic Injury.

OBJECTIVE: Tobacco smoke exposure is a major risk factor for aortic aneurysm development. However, the initial aortic response to tobacco smoke, preceding aneurysm formation, is not well understood. We sought to create a model to determine the effect of solubilized tobacco smoke (STS) on the thoracic and abdominal aorta of mice as well as on cultured human aortic smooth muscle cells (HASMCs). METHODS: Tobacco smoke was solubilized and delivered to mice via implanted osmotic minipumps. Twenty male C57BL/6 mice received STS or vehicle infusion. The descending thoracic, suprarenal abdominal, and infrarenal abdominal segments of the aorta were assessed for elastic lamellar damage, smooth muscle cell phenotype, and infiltration of inflammatory cells. Cultured HASMCs grown in media containing STS were compared to cells grown in standard media in order to verify our in vivo findings. RESULTS: Tobacco smoke solution caused significantly more breaks in the elastic lamellae of the thoracic and abdominal aorta compared to control solution (P< .0001) without inciting an inflammatory infiltrate. Elastin breaks occurred more frequently in the abdominal aorta than the thoracic aorta (P < .01). Exposure to STS-induced aortic microdissections and downregulation of α-smooth muscle actin (α-SMA) by vascular smooth muscle cells (VSMCs). Treatment of cultured HASMCs with STS confirmed the decrease in α-SMA expression. CONCLUSION: Delivery of STS via osmotic minipumps appears to be a promising model for investigating the early aortic response to tobacco smoke exposure. The initial effect of tobacco smoke exposure on the aorta is elastic lamellar damage and downregulation of (α-SMA) expression by VSMCs. Elastic lamellar damage occurs more frequently in the abdominal aorta than the thoracic aorta and does not seem to be mediated by the presence of macrophages or other inflammatory cells.

Cell autonomous TGFß signaling is essential for stem/progenitor cell recruitment into degenerative tendons.

Understanding cell recruitment in damaged tendons is critical for improvements in regenerative therapy. We recently reported that targeted disruption of transforming growth factor beta (TGFß) type II receptor in the tendon cell lineage (Tgfbr2ScxCre) resulted in resident tenocyte dedifferentiation and tendon deterioration in postnatal stages. Here we extend the analysis and identify direct recruitment of stem/progenitor cells into the degenerative mutant tendons. Cre-mediated lineage tracing indicates that these cells are not derived from tendon-ensheathing tissues or from a Scleraxis-expressing lineage, and they turned on tendon markers only upon entering the mutant tendons. Through immunohistochemistry and inducible gene deletion, we further find that the recruited cells originated from a Sox9-expressing lineage and their recruitment was dependent on cell autonomous TGFß signaling. The cells identified in this study thus differ from previous reports of cell recruitment into injured tendons and suggest a critical role for TGFß signaling in cell recruitment, providing insights that may support improvements in tendon repair.

Localized chondro-ossification underlies joint dysfunction and motor deficits in the Fkbp10 mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a genetic disorder that features wide-ranging defects in both skeletal and nonskeletal tissues. Previously, we and others reported that loss-of-function mutations in FK506 Binding Protein 10 (FKBP10) lead to skeletal deformities in conjunction with joint contractures. However, the pathogenic mechanisms underlying joint dysfunction in OI are poorly understood. In this study, we have generated a mouse model in which Fkbp10 is conditionally deleted in tendons and ligaments. Fkbp10 removal substantially reduced telopeptide lysyl hydroxylation of type I procollagen and collagen cross-linking in tendons. These biochemical alterations resulting from Fkbp10 ablation were associated with a site-specific induction of fibrosis, inflammation, and ectopic chondrogenesis followed by joint deformities in postnatal mice. We found that the ectopic chondrogenesis coincided with enhanced Gli1 expression, indicating dysregulated Hedgehog (Hh) signaling. Importantly, genetic inhibition of the Hh pathway attenuated ectopic chondrogenesis and joint deformities in Fkbp10 mutants. Furthermore, Hh inhibition restored alterations in gait parameters caused by Fkbp10 loss. Taken together, we identified a previously unappreciated role of Fkbp10 in tendons and ligaments and pathogenic mechanisms driving OI joint dysfunction.

Reticulocalbin 3 is involved in postnatal tendon development by regulating collagen fibrillogenesis and cellular maturation.

Tendon plays a critical role in the joint movement by transmitting force from muscle to bone. This transmission of force is facilitated by its specialized structure, which consists of highly aligned extracellular matrix consisting predominantly of type I collagen. Tenocytes, fibroblast-like tendon cells residing between the parallel collagen fibers, regulate this specialized tendon matrix. Despite the importance of collagen structure and tenocyte function, the biological mechanisms regulating fibrillogenesis and tenocyte maturation are not well understood. Here we examine the function of Reticulocalbin 3 (Rcn3) in collagen fibrillogenesis and tenocyte maturation during postnatal tendon development using a genetic mouse model. Loss of Rcn3 in tendon caused decreased tendon thickness, abnormal tendon cell maturation, and decreased mechanical properties. Interestingly, Rcn3 deficient mice exhibited a smaller collagen fibril distribution and over-hydroxylation in C-telopeptide cross-linking lysine from α1(1) chain. Additionally, the proline 3-hydroxylation sites in type I collagen were also over-hydroxylated in Rcn3 deficient mice. Our data collectively suggest that Rcn3 is a pivotal regulator of collagen fibrillogenesis and tenocyte maturation during postnatal tendon development.

Tendon and motor phenotypes in the Crtap-/- mouse model of recessive osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is characterized by short stature, skeletal deformities, low bone mass, and motor deficits. A subset of OI patients also present with joint hypermobility; however, the role of tendon dysfunction in OI pathogenesis is largely unknown. Using the Crtap-/- mouse model of severe, recessive OI, we found that mutant Achilles and patellar tendons were thinner and weaker with increased collagen cross-links and reduced collagen fibril size at 1- and 4-months compared to wildtype. Patellar tendons from Crtap-/- mice also had altered numbers of CD146+CD200+ and CD146-CD200+ progenitor-like cells at skeletal maturity. RNA-seq analysis of Achilles and patellar tendons from 1-month Crtap-/- mice revealed dysregulation in matrix and tendon marker gene expression concomitant with predicted alterations in TGF-ß, inflammatory, and metabolic signaling. At 4-months, Crtap-/- mice showed increased αSMA, MMP2, and phospho-NFκB staining in the patellar tendon consistent with excess matrix remodeling and tissue inflammation. Finally, a series of behavioral tests showed severe motor impairments and reduced grip strength in 4-month Crtap-/- mice - a phenotype that correlates with the tendon pathology.

Ezh2 Is Essential for Patterning of Multiple Musculoskeletal Tissues but Dispensable for Tendon Differentiation.

An efficient musculoskeletal system depends on the precise assembly and coordinated growth and function of muscles, skeleton, and tendons. However, the mechanisms that drive integrated musculoskeletal development and coordinated growth and differentiation of each of these tissues are still being uncovered. Epigenetic modifiers have emerged as critical regulators of cell fate differentiation, but so far almost nothing is known about their roles in tendon biology. Previous studies have shown that epigenetic modifications driven by Enhancer of zeste homolog 2 (EZH2), a major histone methyltransferase, have significant roles in vertebrate development including skeletal patterning and bone formation. We now find that targeting Ezh2 through the limb mesenchyme also has significant effects on tendon and muscle patterning, likely reflecting the essential roles of early mesenchymal cues mediated by Ezh2 for coordinated patterning and development of all tissues of the musculoskeletal system. Conversely, loss of Ezh2 in the tendon cells did not disrupt overall tendon structure or collagen organization suggesting that tendon differentiation and maturation are independent of Ezh2 signaling.

Type I and type V procollagen triple helix uses different subsets of the molecular ensemble for lysine posttranslational modifications in the rER.

Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.