Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 integrins in cell--collagen interactions: identification of conformation dependent alpha 1 beta 1 binding sites in collagen type I.

Integrins can mediate the attachment of cells to collagen type I. In the present study we have investigated the possible differences in collagen type I recognition sites for the alpha 1 beta 1 and alpha 2 beta 1 integrins. Different cyanogen bromide (CB) fragments of the alpha 1 (I) collagen chain were used in cell attachment experiments with three rat cell types, defined with regard to expression of collagen binding integrins. Primary rat hepatocytes expressed alpha 1 beta 1, primary rat cardiac fibroblasts alpha 1 beta 1 and alpha 2 beta 1, and Rat-1 cells only alpha 2 beta 1. All three cell types expressed alpha 3 beta 1 but this integrin did not bind to collagen--Sepharose or to immobilized collagen type I in a radioreceptor assay. Hepatocytes and cardiac fibroblasts attached to substrata coated with alpha 1(I)CB3 and alpha 1(I)CB8; Rat-1 cells attached to alpha 1(I)CB3 but only poorly to alpha 1(I)CB8-coated substrata. Cardiac fibroblasts and Rat-1 cells spread and formed beta 1-integrin-containing focal adhesions when grown on substrata coated with native collagen or alpha 1(I)CB3; focal adhesions were also detected in cardiac fibroblasts cultured on alpha 1(I)CB8. The rat alpha 1 specific monoclonal antibody 3A3 completely inhibited hepatocyte attachment to alpha 1(I)CB3 and alpha 1(I)CB8, as well as the attachment of cardiac fibroblasts to alpha 1(I)CB8, but only partially inhibited the attachment of cardiac fibroblasts to alpha 1(I)CB3. 3A3 IgG did not inhibit the attachment of Rat-1 cells to collagen type I or to alpha 1(I)CB3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deficiency of tenascin-X causes a decrease in the level of expression of type VI collagen

Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX-null fibroblasts exhibit decreased cell-matrix and cell-cell adhesion. In this study, we used a differential display technique to determine the genes involved in this process. Differential display analysis of wild-type and TNX-null fibroblasts revealed that mRNA expression level of type VI collagen alpha3 is predominantly decreased in TNX-null fibroblasts. Expression levels of mRNAs of other subunits of type VI collagen, alpha2 and alpha3 chains, were also remarkably decreased in TNX-null fibroblasts. The protein level of alpha3 chain of type VI collagen was also reduced in TNX-null fibroblasts. However, the organization of type VI collagen in the extracellular matrix of TNX-null fibroblasts was similar to that of wild-type fibroblasts. Transient expression of TNX in Balb3T3 cells caused an increase in the level of mRNA of type VI collagen compared with that in vector control and increased the promoter activity of type VI collagen alpha1 subunit gene. In addition, the expression levels of type I collagen and other collagen fibril-associated molecules such as type XII and type XIV collagens, decorin, lumican and fibromodulin in wild-type and TNX-null fibroblasts were compared. It was found that the mRNA expression levels of type I collagen and collagen fibril-associated molecules other than decorin were decreased and that the expression level of decorin was increased in TNX-null fibroblasts. The results suggest the possibility that TNX mediates not only cell-cell and cell-matrix interactions but also fibrillogenesis via collagen fibril-associated molecules.     

Differential effect of hypertonic initiation block on the synthesis of collagen chains by cultured chick embryo cells

The major type of collagen synthesized by fibroblasts or bone cells, type I collagen, consists of two chains normally found in a 2:1 ratio designated alpha 1(I)2 alpha 2(I) or more simply alpha 1(I)2 alpha 2. I have analyzed the relative synthesis of type I chains in these cells under conditions which reduce the initiation of protein synthesis. It was found that in bone cells, which make a large amount of collagen, the alpha 1(I):alpha 2 ratio is unaltered whereas in fibroblasts, which make smaller amounts of collagen, the alpha 2 chain is particularly sensitive to these same conditions. Examination of the collagen secreted into the medium, under these same conditions, also revealed an altered chain ratio from cells making low amounts of collagen.     

Existence of malfunctioning pro alpha2(I) collagen genes in a patient with a pro alpha 2(I)-chain-defective variant of Ehlers-Danlos syndrome

Collagen synthesis was examined in skin fibroblasts from a patient with a variant of Ehlers-Danlos syndrome. The relative rate of collagen synthesis to total protein synthesis in the patient's fibroblasts was always one-half of that in fibroblasts from normal controls. Total collagen synthesis, as assessed by quantification of total hydroxyproline, was also significantly lower than that of controls, indicating that the rate of collagen synthesis by the patient's fibroblasts was decreased compared with that by normal fibroblasts. Analysis of procollagen and collagen components showed the absence of the pro alpha 2(I) chain and its derivatives. Dot-blot and Northern-blot analyses showed the patient's fibroblasts to contain less than 10% of the mRNAs for pro alpha 2(I) found in control fibroblasts. In spite of these results, Southern blot analysis of genomic DNA indicated the presence of the same number of genes for the pro alpha 2(I) collagen chain in the patient's fibroblasts as in control fibroblasts, suggesting malfunctioning pro alpha 2(I) collagen genes as the cause for failure of the patient's fibroblasts to synthesize pro alpha 2(I) collagen chains.     

Human and chick alpha 2(I) collagen mRNA: comparison of the 5' end in osteoblasts and fibroblasts

Type I collagen, a heterotrimer of two alpha 1(I) chains and one alpha 2(I) chain, is the major structural protein of bone, skin, and tendon. The collagen of patients with bone diseases has been studied in skin fibroblasts instead of osteoblasts because the genes for type I collagen are single-copy genes. While these studies should detect structural changes in the gene, they might fail to detect defects in processes which are dependent on tissue-specific expression. The studies reported here sought to determine whether the expression of type I collagen in skin and bone was characterized by the use of alternate promoters or alternative splicing in the N-propeptide region. Primer extension and nuclease S1 protection experiments were used to analyze the structure of the alpha 2(I) mRNA from the 5' end of the gene through the N-telopeptide coding region (exons 1-6) in human and chick osteoblasts and fibroblasts. The protection and primer extension experiments using human and chick mRNA demonstrated identical routes of splicing in skin and bone at the first five splice junctions. These studies provide reassurance that information obtained from the study of type I collagen in fibroblasts may be extrapolated to bone.