SPARC Regulates Processing of Procollagen I and Collagen Fibrillogenesis in Dermal Fibroblasts

Tyler J Rentz,Felicitta Poobalarahi,Paul Bornstein,E Helene Sage,Amy D Bradshaw

doi:10.1074/jbc.m700167200

Abstract

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 wild-type 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.

Highlights

Expression of SPARC is elevated during development and decreases upon differentiation in a majority of tissues [4]
We have reported that collagen fibrils in adult SPARC-null skin were smaller and more uniform in diameter than those of WT mice [12]
We sought to determine whether the absence of SPARC was associated with phenotypic differences in collagen fibril morphology in the dermis during times characterized by active collagen fibril aggregation

Summary

EXPERIMENTAL PROCEDURES

Reagents—Cell culture reagents were from Invitrogen. rSPARC was generated by baculovirus infection of insect cells and purified as described in Ref. 15. Analysis of ECM Production and Deposition—Primary dermal fibroblasts plated at equal concentrations were labeled with 25 ␮Ci/ml [2,3,4,5-3H]proline (PerkinElmer Life Sciences) in growth media for 18 –24 h to assess collagen production in media and cell layers from WT and SPARC-null cells. Immunoblot analysis was performed by transfer of separated proteins to nitrocellulose and detection with anti-murine collagen I antibodies. Labeled conditioned media generated by equal numbers of WT and SPARC-null cells grown in ascorbate for 18 –24 h were incubated with an anti-N-propeptide rabbit polyclonal antibody against recombinant mouse N-propeptide that was produced in insect cells, or polyclonal anti-murine SPARC antibodies (R&D Systems, Minneapolis, MN). The pulse labeling media were removed, and the cell layer was rinsed in phosphate-buffered saline followed by incubation in chase media (growth media with ascorbate and 1 mg/ml unlabeled proline) for the indicated time periods. Coverslips were mounted in Anti-Fade reagent (Molecular Probes, Eugene, OR) and were viewed on a Leica microscope equipped for epifluorescence

RESULTS

Quantification from three separate experiments is shown in

DISCUSSION