Dataset Information

Collagens VI and XII form complexes mediating osteoblast interactions during osteogenesis.

ABSTRACT: Bone formation is precisely regulated by cell-cell communication in osteoblasts. We have previously demonstrated that genetic deletion of Col6a1 or Col12a1 impairs osteoblast connections and/or communication in mice, resulting in bone mass reduction and bone fragility. Mutations of the genes encoding collagen VI cause Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM), which have overlapping phenotypes involving connective tissue and muscle. Recent studies have identified COL12A1 gene mutations in patients with UCMD- and BM-like disorders harboring no COL6 mutations, indicating the shared functions of these collagens in connective tissue homeostasis. The purpose of this investigation has been to test the hypothesis that collagens VI and XII have coordinate regulatory role(s) during bone formation. We analyzed the localization of collagens VI and XII relative to primary osteoblasts during osteogenesis. Immunofluorescence analysis demonstrated that collagens VI and XII colocalized in matrix bridges between adjacent cells during periods when osteoblasts were establishing cell-cell connections. Quantification of cells harboring collagen bridges demonstrated that matrix bridges were composed of collagens VI and XII but not collagen I. Interestingly, matrix bridge formation was impaired in osteoblasts deficient in either Col6a1 or Col12a1, suggesting that both collagens were indispensable for matrix bridge formation. These data demonstrate, for the first time, a functional relationship between collagens VI and XII during osteogenesis and indicate that a complex containing collagens VI and XII is essential for the formation of a communicating cellular network during bone formation.

SUBMITTER: Izu Y

PROVIDER: S-EPMC4875952 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Rationale: Angiogenesis and osteogenesis are highly coupled processes which are indispensable to bone repair. However, the underlying mechanism(s) remain elusive. To bridge the gap in understanding the coupling process is crucial to develop corresponding solutions to abnormal bone healing. Epidermal growth factor-like protein 6 (EGFL6) is an angiogenic factor specifically and distinctively up-regulated during osteoblast differentiation. In contrast with most currently known osteoblast-derived coupling factors, EGFL6 is highlighted with little or low expression in other cells and tissues. Methods: In this study, primary bone marrow mesenchymal stem cells (MSCs) and osteoblastic cell line (MC3T3-E1) were transduced with lentiviral silencing or overexpression constructs targeting EGFL6. Cells were induced by osteogenic medium, followed by the evaluation of mineralization as well as related gene and protein expression. Global and conditional knockout mice were established to examine the bone phenotype under physiological condition. Furthermore, bone defect models were created to investigate the outcome of bone repair in mice lacking EGFL6 expression. Results: We show that overexpression of EGFL6 markedly enhances osteogenic capacity in vitro by augmenting bone morphogenic protein (BMP)-Smad and MAPK signaling, whereas downregulation of EGFL6 diminishes osteoblastic mineralization. Interestingly, osteoblast differentiation was not affected by the exogenous addition of EGFL6 protein, thereby indicating that EGFL6 may regulate osteoblastic function in an intracrine manner. Mice with osteoblast-specific and global knockout of EGFL6 surprisingly exhibit a normal bone phenotype under physiological conditions. However, EGFL6-deficiency leads to compromised bone repair in a bone defect model which is characterized by decreased formation of type H vessels as well as osteoblast lineage cells. Conclusions: Together, these data demonstrate that EGFL6 serves as an essential regulator to couple osteogenesis to angiogenesis during bone repair.

Project description:Osteogenesis imperfecta (OI) types V and VI are caused, respectively, by a unique dominant mutation in IFITM5, encoding BRIL, a transmembrane ifitm-like protein most strongly expressed in the skeletal system, and recessive null mutations in SERPINF1, encoding pigment epithelium-derived factor (PEDF). We identified a 25-year-old woman with severe OI whose dermal fibroblasts and cultured osteoblasts displayed minimal secretion of PEDF, but whose serum PEDF level was in the normal range. SERPINF1 sequences were normal despite bone histomorphometry consistent with type VI OI and elevated childhood serum alkaline phosphatase. We performed exome sequencing on the proband, both parents, and an unaffected sibling. IFITM5 emerged as the candidate gene from bioinformatics analysis, and was corroborated by membership in a murine bone co-expression network module containing all currently known OI genes. The de novo IFITM5 mutation was confirmed in one allele of the proband, resulting in a p.S40L substitution in the intracellular domain of BRIL but was absent in unaffected family members. IFITM5 expression was normal in proband fibroblasts and osteoblasts, and BRIL protein level was similar to control in differentiated proband osteoblasts on Western blot and in permeabilized mutant osteoblasts by microscopy. In contrast, SERPINF1 expression was decreased in proband osteoblasts; PEDF was barely detectable in conditioned media of proband cells. Expression and secretion of type I collagen was similarly decreased in proband osteoblasts; the expression pattern of several osteoblast markers largely overlapped reported values from cells with a primary PEDF defect. In contrast, osteoblasts from a typical case of type V OI, with an activating mutation at the 5'-terminus of BRIL, have increased SERPINF1 expression and PEDF secretion during osteoblast differentiation. Together, these data suggest that BRIL and PEDF have a relationship that connects the genes for types V and VI OI and their roles in bone mineralization.

Project description:Osteoblast-derived extracellular vesicles (EV) are a collection of secreted (sEVs) and matrix-bound nanoparticles that function as foci for mineral nucleation and accumulation. Due to the fact sEVs can be isolated directly from the culture medium of mineralizing osteoblasts, there is growing interest their application regenerative medicine. However, at present therapeutic advancements are hindered by a lack of understanding of their precise temporal contribution to matrix mineralization. This study advances current knowledge by temporally aligning sEV profile and protein content with mineralization status. sEVs were isolated from mineralizing primary osteoblasts over a period of 1, 2, and 3 weeks. Bimodal particle distributions were observed (weeks 1 and 3: 44 and 164 nm; week 2: 59 and 220 nm), indicating a heterogeneous population with dimensions characteristic of exosome- (44 and 59 nm) and microvesicle-like (164 and 220 nm) particles. Proteomic characterization by liquid chromatography tandem-mass spectrometry (LC-MS/MS) revealed a declining correlation in EV-localized proteins as mineralization advanced, with Pearson correlation-coefficients of 0.79 (week 1 vs. 2), 0.6 (2 vs. 3) and 0.46 (1 vs. 3), respectively. Principal component analysis (PCA) further highlighted a time-dependent divergence in protein content as mineralization advanced. The most significant variations were observed at week 3, with a significant (p < 0.05) decline in particle concentration, visual evidence of EV rupture and enhanced mineralization. A total of 116 vesicle-localized proteins were significantly upregulated at week 3 (56% non-specifically, 19% relative to week 1, 25% relative to week 2). Gene ontology enrichment analysis of these proteins highlighted overrepresentation of genes associated with matrix organization. Of note, increased presence of phospholipid-binding and calcium channeling annexin proteins (A2, A5, and A6) indicative of progressive variations in the nucleational capacity of vesicles, as well as interaction with the surrounding ECM. We demonstrate sEV-mediated mineralization is dynamic process with variations in vesicle morphology and protein content having a potential influence on developmental changes matrix organization. These findings have implications for the selection and application of EVs for regenerative applications.

Project description:BackgroundThe vitreoretinal interface is the border of the cortical vitreous and the inner surface of the retina. The adhesion of the cortical vitreous to the ILM, namely vitreoretinal adhesion, involves a series of complex molecular adhesion mechanisms and has been considered as an important pathogenic factor in many vitreoretinal diseases. The presence of type VI collagen at the vitreoretinal interface and its possible interaction with collagens and glycoproteins indicates that type VI collagen may contribute to the vitreoretinal adhesion.PurposeTo clarify the ultrastructural location of type VI collagen and its relationship to type II and IV collagens at the vitreoretinal interface.MethodsThe ultrastructural localization of type II, IV and VI collagens in the adult human vitreoretinal interface of five donor eyes was evaluated by transmission electron microscopy using immunogold labeling.ResultsIn the pre-equatorial region, we observed densely packed vitreous lamellae with a partly intraretinal course containing type II and VI collagens, reticular structures containing type IV and VI collagens and a thin inner limiting membrane (ILM) containing type IV and VI collagens in a linear distribution pattern. From the anterior to the posterior retina, the linear pattern of type IV and VI collagen labeling gradually became more diffusely present throughout the entire thickness of the ILM.ConclusionsThe presence of type VI collagen in vitreous lamellae penetrating the ILM into the superficial retina suggests that type VI collagen may be involved in the organization of vitreous fibers into lamellae and in the adhesion of the vitreous fibers to the retina. The close relation of type VI to type IV collagen in the ILM suggests that type VI collagen is an important collagen type in the ILM. The topographic variations of type IV and VI collagens in the different regions of the ILM suggest a regional heterogeneity of the ILM. The reticular labeling pattern of type IV and VI collagens observed in the anterior vitreous are highly similar to labeling patterns of blood vessel walls. In the anterior vitreous, they may represent remnants of the regressed embryonic hyaloid blood vessel system. Their presence is in support of the theory on interactive remodeling of the developing vitreous as opposed to the main stream theory of displacement and compression of the primary by the secondary vitreous.

Dataset Information

Collagens VI and XII form complexes mediating osteoblast interactions during osteogenesis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets