Tensile stress-dependent collagen XII and fibronectin production by fibroblasts requires separate pathways.
The intracellular mechanisms controlling mechano-dependent production of the two extracellular matrix proteins collagen XII and fibronectin were analyzed. Fibroblasts were cultured on either tensed (attached) or released (floating) collagen type-I gels, respectively. Collagen XII and fibronectin production was three- to fivefold higher under tensed than under released conditions. The general inhibitor of tyrosine phosphorylation, genistein (50 microM), and the MAP kinase inhibitor PD98059 (20 microM) selectively reduced collagen XII accumulation by tensed cultures. Addition of PD98059, but not genistein, downregulated tensile stress-induced tyrosine phosphorylation levels of ERK1/2 and focal adhesion kinase. Staurosporine as well as pretreatment with phorbol ester, which constitute means to downregulate classical and novel PKC activity, specifically blocked collagen XII but not fibronectin accumulation in tensed fibroblasts. ERK1/2 phosphorylation levels were not affected by staurosporine treatment. Chronic exposure to the protein kinase C inhibitors bisindolylmaleimide and calphostin C blocked increased production of both fibronectin and collagen XII from cells under tension. The data manifest that the mechano-dependent production of collagen XII and fibronectin requires separate pathways. The FAK-ERK1/2 pathway, a genistein-sensitive tyrosine kinase, and a distinct classical/novel PKC appear selectively required for increased production of collagen XII in cells under tensile stress, whereas fibronectin induction is regulated by a different PKC-dependent pathway. (+info)
Differential expression of type XII collagen in developing chicken metatarsal tendons.
Type XII collagen is a fibril-associated collagen with multiple functional domains. The purpose of this work was to determine its role in regulating tendon matrix assembly. The temporal and spatial expression patterns of both collagen and mRNA were analysed in developing chicken metatarsal tendons using immunofluorescence microscopy, in situ hybridization and real-time quantitative PCR. Temporally, type XII collagen was present during all stages of development (day 14-hatch). However, spatially, type XII collagen expression shifted from the entire tendon at day 14, when the tendon is immature and fascicles are not well developed, to the interfacial matrix (endotendinium) associated with developing fascicles. This shift was obvious beginning at day 17, becoming prominent at day 19. Associated with this shift was a gradual decrease in type XII collagen reactivity in the tendon proper (non-sheath). By hatching, the reactivity was sequestered almost exclusively to the sheaths with some reactivity remaining at the fibroblast-matrix interface within the fascicle. In situ hybridization indicated that fibroblasts in the tendon expressed type XII collagen mRNA homogeneously at day 14. However, by hatching, when the tendon matures, type XII collagen is restricted primarily to the sheath cells. Quantitative PCR analyses, of NC3 splice variants, demonstrated highest expression levels for the short splice variant mRNA at days 14-17, followed by a significant decrease at day 19 with levels remaining constant to adult. Long variant mRNA expression was highest at day 14 then decreased and was constant from day 17 to adult. These changing patterns may be related to the spatial shift in type XII collagen expression to the sheaths. Differential temporal and spatial expression patterns indicate that type XII collagen functions to integrate the developing tendon matrices and fascicles into a functional unit. (+info)
Expression of FACIT collagens XII and XIV during bleomycin-induced pulmonary fibrosis in mice.
Collagens XII and XIV are members of a subfamily of fibril-associated collagens with interrupted triple-helices (FACITs) that facilitate the interactions of adjacent collagen fibrils. Using immunohistochemistry and in situ hybridization, we analyzed the spatial and temporal expression pattern of collagens XII and XIV during bleomycin-induced pulmonary fibrosis. C57Bl mice were treated with bleomycin (1 U, i.p., every other day for 8 days) or saline (control), and lung tissue samples were analyzed 2-12 weeks later. Collagen I protein expression was increased in the lung 2 weeks post bleomycin treatment and persisted for at least 12 weeks. In contrast, collagen XII and XIV expression was low until 4 weeks after bleomycin treatment. Whereas collagen XII expression was greatest between 4 weeks and 8 weeks, expression of collagen XIV persisted from 4 to 12 weeks, which suggests that these two proteins may play distinct roles in the fibrotic process. The mRNA for lysyl oxidase (LOX), an enzyme for cross-linking of collagens, had a delayed increase in the lung after bleomycin administration. It reached a maximum after 8 weeks, and persisted throughout the 12 weeks of the study. These data support the hypothesis that fibrosis is a multistep process that involves both collagen accumulation and changes in the molecules that modulate the biomechanical properties of fibrils. (+info)
Collagen XII interacts with avian tenascin-X through its NC3 domain.
Large oligomeric proteins often contain several binding sites for different molecules and can therefore induce formation of larger protein complexes. Collagen XII, a multidomain protein with a small collagenous region, interacts with fibrillar collagens through its C-terminal region. However, no interactions to other extracellular proteins have been identified involving the non-collagenous N-terminal NC3 domain. To further elucidate the components of protein complexes present close to collagen fibrils, different extracellular matrix proteins were tested for interaction in a solid phase assay. Binding to the NC3 domain of collagen XII was found for the avian homologue of tenascin-X that in humans is linked to Ehlers-Danlos disease. The binding was further characterized by surface plasmon resonance spectroscopy and supported by immunohistochemical co-localization in chick and mouse tissue. On the ultrastructural level, detection of collagen XII and tenascin-X by immunogold labeling confirmed this finding. (+info)
Extracellular matrix gene alternative splicing by trabecular meshwork cells in response to mechanical stretching.
PURPOSE: Elevated intraocular pressure (IOP), sensed as mechanical stretching by trabecular meshwork (TM) cells, triggers extracellular matrix (ECM) remodeling. In addition to changes in gene expression, alternative mRNA splicing may alter ECM protein isoforms. Changes in mRNA expression and alternative splicing of four ECM molecules in response to mechanical stretching of TM cells were investigated. METHODS: Porcine TM cells were mechanically stretched for 12, 24, or 48 hours. RNA was isolated, and RT-PCR was performed with primers that flanked alternatively spliced domains. PCR products were identified by DNA sequencing. Quantitative RT-PCR (qRT-PCR) was performed with primers positioned within nonspliced and spliced regions of the genes. RESULTS: Total levels of tenascin C, collagen type XII, and CD44 mRNA were increased, whereas versican mRNA levels were decreased in response to the mechanical stretch. In addition, each of these genes expressed alternate mRNA isoforms. Transcripts containing the fibronectin type III domain D of tenascin C, the long NC3 isoform of collagen type XII, the V1 isoform of versican, and exons v7 and v8 of CD44 all increased in response to mechanical stretching. A novel isoform of collagen type XII was observed that resulted in deletion of two exons, a frameshift, and a premature stop codon. This isoform was expressed only by stretched TM cells. CONCLUSIONS: These alternative splicing events led to the modulation of potential GAG attachment sites and other ECM-binding motifs. These changes should affect TM cell-ECM and/or protein-protein interactions during the ECM remodeling that occurs coincident with homeostatic restoration of IOP to normal. (+info)
Mechanical strain increases expression of type XII collagen in murine osteoblastic MC3T3-E1 cells.
In adult mouse, the mRNA corresponding to the alpha1 chain of type XII collagen (alpha 1(XII)) is predominantly detected in the bone. Additionally, murine osteoblastic cells, MC3T3-E1, increased the mRNA level of alpha 1(XII) response to the mechanical strain in the stretch culture system. Cyclic stretch stress resulted in a threefold increase in mRNA level of alpha 1(XII) as compared to the control experiment in MC3T3-E1. Transient transfection assays employing a reporter construct, together with site-directed mutagenesis studies, suggested that the AP-1 binding site in the first exon of mouse alpha 1(XII) gene is important for stretch stress-mediated upregulation of alpha 1(XII) expression. Electrophoretic mobility shift assay and associated antibody supershift experiments showed that stretch stress promotes the binding of c-Jun and JunD. Further chromatin immunoprecipitation experiments confirmed the participation of these transcription factors in the region. Also, the exogenous induction of the dominant negative form of c-Jun canceled the effect of stretch stress on the stimulation of the alpha 1(XII) gene. Here, we reported a potential responsive element to the stretch stress in mouse alpha 1(XII) gene. These data will provide new information on the mechanical strain-mediated transcriptional control of alpha 1(XII)-mediated fibrillogenesis in the bone. (+info)
Tibolone inhibits bone resorption without secondary positive effects on cartilage degradation.