A characterization of the factors that control collagen fibril formation is critical for an understanding of tissue organization and the mechanisms that lead to fibrosis. SPARC (secreted protein acidic and rich in cysteine) is a counter-adhesive protein that binds collagens. Herein we show that collagen fibrils in SPARC-null skin from mice 1 month of age were inefficient in fibril aggregation and accumulated in the diameter range of 60 -70 nm, a proposed intermediate in collagen fibril growth. In vitro, procollagen I produced by SPARC-null dermal fibroblasts demonstrated an initial preferential association with cell layers, in comparison to that produced by wild-type fibroblasts. However, the collagen I produced by SPARC-null cells was not efficiently incorporated into detergent-insoluble fractions. Coincident with an initial increase in cell association, greater amounts of total collagen I were present as processed forms in SPARC-null versus wild-type cells. Addition of recombinant SPARC reversed collagen I association with cell layers and decreased the processing of procollagen I in SPARC-null cells. Although collagen fibers formed on the surface of SPARC-null fibroblasts earlier than those on wildtype cells, fibers on SPARC-null fibroblasts did not persist. We conclude that SPARC mediates the association of procollagen I with cells, as well as its processing and incorporation into the extracellular matrix.Matricellular proteins are defined as proteins that are associated with the extracellular matrix (ECM) 2 but are not considered structural components of the ECM, in contrast to classical ECM proteins such as laminin and collagen I (1, 2). SPARC is a prototypic matricellular protein that exhibits counter-adhesive and anti-proliferative activity when added to cultured cells (3). SPARC has been shown to bind to a number of ECM proteins including collagens I, III, and IV (3).Expression of SPARC is elevated during development and decreases upon differentiation in a majority of tissues (4). However, expression of SPARC persists in tissues in which ECM remodeling is ongoing, such as bone and gut epithelia (4). Increased levels of SPARC are detected in response to injury where ECM remodeling is initiated, with fibroses in liver, lungs, and kidney, and in the skin of individuals with scleroderma (5-8). Hence, SPARC expression patterns implicate this protein as an important mediator of collagen I deposition and/or remodeling.The ␣1(I) and ␣2(I) subunits of procollagen I are synthesized with N-and C-propeptides that are enzymatically released by specific proteases to yield processed collagen I (9). Processing of procollagen I to collagen I is essential for correct assembly of collagen fibrils. Spatial and temporal regulation of procollagen processing has been proposed as a potential regulatory event in collagen fibril assembly (9). For example, antibodies against the N-propeptide of collagen I were immunolocalized exclusively to the surface of collagen fibrils that were 20 -40 nm in diameter (10). Retention of the prop...

Myofibroblasts are a differentiated fibroblast cell type characterized by increased contractile capacity and elevated production of extracellular matrix (ECM) proteins. In the heart, myofibroblast expression is implicated in fibrosis associated with pressure-overload hypertrophy, among other pathologies. Although enhanced expression of ECM proteins by myofibroblasts is established, few studies have addressed the nature of the ECM deposited by myofibroblasts. To characterize ECM production and assembly by cardiac myofibroblasts, we developed a threedimensional (3D) culture system using primary cardiac fibroblasts seeded into a nylon mesh that allows us to reversibly interconvert between myofibroblast and fibroblast phenotypes. We report that an increase in collagen I production by myofibroblasts was accompanied by a significant increase in collagen deposition into insoluble ECM. Furthermore, myofibroblasts exhibited increased levels of procollagen ␣1(I) with C-propeptide attached (and N-propeptide removed) relative to procollagen ␣1(I) compared with fibroblast cultures. An increase in production of the myofibroblast-associated splice variant of fibronectin (EDA-Fn) was seen in myofibroblast 3D cultures. Because the regulation of procollagen I processing is known to have profound effects on ECM assembly, differences in procollagen I secretion and maturation coupled with expression of EDA-Fn are shown to contribute to the production of a distinct ECM by the cardiac myofibroblast.

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