Absence of feedback regulation in the synthesis of COL1A1
Introduction
The extracellular matrix (ECM) is a three-dimensional (3D) scaffold of collagen, fibronectin, and several other proteins (Green and Yamada, 2007, Serebriiskii et al., 2008, Ozbek et al., 2010). The ECM promotes signalling, provides structural support to cells and serves as a reservoir for growth factors, cytokines and enzymes (Cukierman, 2002). It is deposited by several cell types to create an environment best suited for their function. Fibroblasts and other mesenchymal cells are important cells involved in ECM remodelling. ECM remodelling occurs during processes such as angiogenesis, wound repair and embryogenesis (Prockop and Kivirikko, 1984, Beacham and Cukierman, 2005, Tucker and Chiquet-Ehrismann, 2009). Limited ECM turnover occur in normal adult tissues whilst in many pathological conditions the fine balance between ECM synthesis and degradation is disrupted. Excessive ECM destruction is associated with many diseases such as rheumatoid arthritis, periodontitis and tumour invasion and metastasis (Forget et al., 1999, Curran and Murray, 1999, Chiquet-Ehrismann and Chiquet, 2003). Excessive deposition of the ECM results in diseases such as fibrosis, systemic sclerosis and scleroderma (Trojanowska et al., 1998, Bornstein and Sage, 1989).
Although the use of reconstituted purified ECM proteins and their gels have provided many valuable insights into the roles of three-dimensionality, matrix tension and elasticity in cell function (Bissell et al., 2002, Hoshiba et al., 2006), the 3D microenvironments in living animals contain a variety of ECM proteins assembled in a defined ratio. A better approach beyond the use of reconstituted ECM involves using natural, cell-derived 3D ECM such as fibroblast-derived ECM (fd-ECM) (Cukierman, 2002, Cukierman, 2005, Castello-Cros and Cukierman, 2009). These matrices are highly fibrillar and contain imprints originally occupied by the cells that generated the matrix. Cultivation of cells on a layer of ECM proteins such as collagen and fibronectin is known to result in increased extracellular protease activity (Hanagata et al., 2006). These proteases are mostly matrix metalloproteinases (MMPs) (Phillips and Bonassar, 2005, Bjőrklund and Koivunen, 2005). Tissue inhibitors of matrix metalloproteinases (TIMPs) vary in their ability to inhibit different MMPs. Another family of proteases is the cathepsins that are mainly intracellular proteases. Cathepsins activity is regulated by various inhibitors of the cystatin super family. Cystatins A and B are the main intracellular inhibitors, with cystatin C acting primarily extracellularly. The balance between the levels of MMPs, TIMPs, cathepsins and cystatins is tightly regulated and may be swayed by changes in the microenvironment (Laliberte et al., 2001, Helary et al., 2005). Collagen degradation occurs through both intracellular and extracellular pathways, with few proteases capable of degrading native collagen (Laliberte et al., 2001, Holmbeck et al., 1999).
The predominant ECM component, type I collagen, is a product of two genes, COL1A1 and COL1A2. These two genes are regulated in a species and tissue specific manner (Stefanovic, 2005, Alexakis et al., 2006). In this study we investigated the mechanism by which the fd-ECM regulates COL1A1 gene expression. This is fundamentally important in the physiological remodelling of connective tissues and could be utilised in the pathological degradation of collagen.
Section snippets
Materials
Pepsin was obtained from Southern Biotech (Birmingham, AL). SDS-PAGE molecular weight standards were from Bio-Rad (Philadelphia, Pennsylvania). Purified type I collagen was purchased as a solution of acid-stabilized monomer (Vitrogen; Cohesion Technologies, Inc., Palo Alto, CA). Purified fibronectin and TGF-β were from Sigma (St. Louis, MO). TAPI-1 was purchased from Merck Biosciences (Darmstadt, Germany). Ammonium hydroxide was purchased from Merck Biosciences (Darmstadt, Germany).
Cell culture and preparation of fd-ECM
A human lung
Fibroblast-derived ECM reduces the expression of type I collagen
Type I collagen production in fibroblasts grown on a pre-formed fd-ECM was investigated by examining both intracellular and secreted type I collagen levels. Medium proteins were digested with pepsin and since triple helical type I collagen is resistant to pepsin, it remains intact whilst practically all other proteins are digested. Intracellular type I collagen was detected with anti-type I collagen antibody. We confirmed previous studies that showed that the levels of both intracellular and
Discussion
Remodelling of collagen by fibroblasts is a fundamentally important physiological process that allows connective tissues to replace old and damaged fibres and to adapt to changing functional demands. Fibroblasts detect any changes in their environment and translate this information into an adaptive response such as either an increase or a decrease in ECM protein production (Cukierman, 2002, Beacham and Cukierman, 2005). Collagen matrices, gels, and other synthetic support systems have been used
Conclusion
Based on the results presented here and together with our published results (Dzobo et al., 2012) we propose that growth of fibroblasts on a fd-ECM results in downregulation of COL1A2 gene transcription, whilst COL1A1 gene transcription is unaffected. The α2(1) polypeptide chain becomes limiting in the formation of type I collagen resulting in decreased levels of type I collagen formation, excess α1(1) polypeptide chains are then degraded through the action of intracellular and extracellular
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
This work is based upon the research supported by the International Centre for Genetic Engineering and Biotechnology (ICGEB), the South African Medical Research Council and the University of Cape Town. We would like to thank Ellen Ngarande and Zenaria Abbas for the technical assistance.
The funders had no role in the conduct of the research or the preparation of the manuscript.
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2021, Journal of Biological ChemistryCitation Excerpt :Although the disease variants responsible for developing OI were different, osteocytes of oim and Crtap mice also demonstrated overexpression of Col1a1 and Col1a2 (34). Since a feedback regulation of type I collagen via α2β1 integrin has been reported (35, 36), impaired ECM formation may cause the excessive production of type I collagen. Because some of the type I collagen produced excessively may have had structural defectiveness due to a glycine substitution mutation, there was difficulty in their assembly and deposition on the culture dish (Fig. 3C), and ECM containing mutant type I collagen might be associated with the excessive production of type I collagen (Fig. 4C), resulting in a vicious circle.