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

Rentz, Tyler J.; Poobalarahi, Felicitta; Börnstein, Paul; Sage, E. Helene; Bradshaw, Amy D.

doi:10.1074/jbc.m700167200

Cited by 148 publications

(146 citation statements)

References 49 publications

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“…Proteins were transferred to nitrocellulose and blocked with 1% casein, and membranes were probed with rabbit anti-mouse collagen I (MD Biosciences). The anti-collagen I antibody recognizes the procollagen ␣1(I) chain with the N-and C-propeptides, the pC-propeptide chain (pC-␣1(I)), and the ␣1(I) chain with both the N-and C-propeptides cleaved (42,43). Membranes were washed with TBS-T (10 mM Tris-HCl, pH 7.6, 100 mM NaCl, and 0.1% Tween 20), probed with appropriate secondary antibody, and developed using Western Lightning MEFs.…”

Section: Methodsmentioning

confidence: 99%

Intracellular Calreticulin Regulates Multiple Steps in Fibrillar Collagen Expression, Trafficking, and Processing into the Extracellular Matrix

Graham

Sweetwyne

Pallero

et al. 2010

Journal of Biological Chemistry

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Calreticulin (CRT), a chaperone and Ca2؉ regulator, enhances wound healing, and its expression correlates with fibrosis in animal models, suggesting that CRT regulates production of the extracellular matrix. However, direct regulation of collagen matrix by CRT has not been previously demonstrated. We investigated the role of CRT in the regulation of fibrillar collagen expression, secretion, processing, and deposition in the extracellular matrix by fibroblasts. Mouse embryonic fibroblasts deficient in CRT (CRT ؊/؊ MEFs) have reduced transcript levels of fibrillar collagen I and III and less soluble collagen as compared with wild type MEFs. Correspondingly, fibroblasts engineered to overexpress CRT have increased collagen type I transcript and protein. Collagen expression appears to be regulated by endoplasmic reticulum (ER) calcium levels and intracellular CRT, because thapsigargin treatment reduced collagen expression, whereas addition of exogenous recombinant CRT had no effect. CRT ؊/؊ MEFs exhibited increased ER retention of collagen, and collagen and CRT were co-immunoprecipitated from isolated cell lysates, suggesting that CRT is important for trafficking of collagen through the ER. CRT ؊/؊ MEFs also have reduced type I procollagen processing and deposition into the extracellular matrix. The reduced collagen matrix deposition is partly a consequence of reduced fibronectin matrix formation in the CRT-deficient cells. Together, these data show that CRT complexes with collagen in cells and that CRT plays critical roles at multiple stages of collagen expression and processing. These data identify CRT as an important regulator of collagen and suggest that intracellular CRT signaling plays an important role in tissue remodeling and fibrosis.Calreticulin (CRT), 3 also known as the C1q receptor, is an endoplasmic reticulum (ER) chaperone, and a modulator of intracellular calcium signaling. CRT also is a multifunctional protein that is present in numerous cellular compartments, including the ER, cytoplasm, nucleus, at the cell surface, and as a released protein (1-5). There is evidence to suggest the involvement of CRT in tissue remodeling and wound healing. In a porcine dermal wound healing model, topical application of purified CRT increases the rate of wound healing and wound tensile strength (6). Proteomic data show up-regulation of CRT expression with fibrosis in a rat model of unilateral ureteric obstruction kidney fibrosis and in a mouse model of bleomycininduced lung fibrosis (7). The mechanisms by which CRT regulates tissue remodeling are not well understood, although fibronectin matrix deposition and modulation of cell adhesion, motility, proliferation, and matrix metalloproteinase expression have been implicated (6, 8 -13).CRT has effects on the extracellular matrix (ECM) and cellular responses to the ECM. Fibroblasts overexpressing CRT have increased fibronectin mRNA, protein, and matrix deposition, and cells lacking CRT express less fibronectin than wild type cells (9,14). CRT in the ER is thought to reg...

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Section: Methodsmentioning

confidence: 99%

Intracellular Calreticulin Regulates Multiple Steps in Fibrillar Collagen Expression, Trafficking, and Processing into the Extracellular Matrix

Graham

Sweetwyne

Pallero

et al. 2010

Journal of Biological Chemistry

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“…SPARC is essential for embryo development in invertebrates (16,17) and has been suggested to act as a chaperone for basement membrane collagen IV (17). Mice lacking SPARC develop early onset cataract, lax skin and bone loss; this phenotype is likely to result, at least in part, from perturbed collagen fibrillogenesis or cell adhesion (14,15). Human SPARC consists of an acidic 52-residue segment followed by a follistatin-like (FS) domain and an ␣-helical domain (EC) containing 2 unusual calcium-binding EF-hands and the collagen-binding site (18)(19)(20).…”

mentioning

confidence: 99%

“…SPARC (also called osteonectin or BM-40) is a small evolutionarily conserved glycoprotein that modulates cell-matrix interactions and collagen assembly (14,15). SPARC is essential for embryo development in invertebrates (16, 17) and has been suggested to act as a chaperone for basement membrane collagen IV (17).…”

mentioning

confidence: 99%

“…The atomic details of this important interaction were revealed by a crystal structure of the integrin ␣2 I-domain bound to a 21-residue triple helical peptide, providing the only example to date of a vertebrate protein-collagen complex (10). More recent studies have identified a GVMGFO motif, conserved in collagens I-III and situated Ϸ100 residues (Ϸ30 nm) upstream of the GFOGER motif, as a binding hotspot for 3 structurally and functionally distinct proteins: the plasma protein von Willebrand factor (VWF), whose interaction with collagen contributes to haemostasis (11); the receptor tyrosine kinase DDR2 (12); and the extracellular matrix protein SPARC (13).SPARC (also called osteonectin or BM-40) is a small evolutionarily conserved glycoprotein that modulates cell-matrix interactions and collagen assembly (14,15). SPARC is essential for embryo development in invertebrates (16, 17) and has been suggested to act as a chaperone for basement membrane collagen IV (17).…”

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confidence: 99%

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Structural basis of sequence-specific collagen recognition by SPARC

Hohenester

Sasaki

Giudici

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

124

115

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Protein interactions with the collagen triple helix play a critical role in collagen fibril formation, cell adhesion, and signaling. However, structural insight into sequence-specific collagen recognition is limited to an integrin-peptide complex. A GVMGFO motif in fibrillar collagens (O denotes 4-hydroxyproline) binds 3 unrelated proteins: von Willebrand factor (VWF), discoidin domain receptor 2 (DDR2), and the extracellular matrix protein SPARC/osteonectin/BM-40. We report the crystal structure at 3.2 Å resolution of human SPARC bound to a triple-helical 33-residue peptide harboring the promiscuous GVMGFO motif. SPARC recognizes the GVMGFO motifs of the middle and trailing collagen chains, burying a total of 720 Å 2 of solvent-accessible collagen surface. SPARC binding does not distort the canonical triple helix of the collagen peptide. In contrast, a critical loop in SPARC is substantially remodelled upon collagen binding, creating a deep pocket that accommodates the phenylalanine residue of the trailing collagen chain (''Phe pocket''). This highly restrictive specificity pocket is shared with the collagenbinding integrin I-domains but differs strikingly from the shallow collagen-binding grooves of the platelet receptor glycoprotein VI and microbial adhesins. We speculate that binding of the GVMGFO motif to VWF and DDR2 also results in structural changes and the formation of a Phe pocket. extracellular matrix ͉ X-ray crystallography C ollagen, the most abundant protein in vertebrates, is characterized by a triple-helical structure of 3 polypeptide chains containing repetitive glycine-X-XЈ triplets; the X and XЈ positions, respectively, are often occupied by the imino acids proline and 4-hydroxyproline (O). The 28 human collagens have numerous essential functions in tissue formation, stability and homeostasis, and mutations in collagen genes cause many human diseases (1). Collagens I-III, V and XI form supramolecular fibrils that lend mechanical stability to the vertebrate body. Normal collagen fibrillogenesis in vivo requires a number of globular proteins interacting with the triple helix, such as small leucine-rich repeat proteoglycans (2). Cellular interactions with collagen are mediated by a diverse group of transmembrane collagen receptors, including integrins, discoidin domain receptors (DDRs) and members of the Ig superfamily (3-5). Although the structure of the collagen triple helix has been known for Ͼ50 years (6), protein-collagen interactions remain poorly understood at the atomic level.Synthetic triple-helical peptides have been invaluable in mapping specific binding sites within collagen (7). The major integrin-binding site in fibrillar collagens I and II is a GFOGER motif (8, 9). The atomic details of this important interaction were revealed by a crystal structure of the integrin ␣2 I-domain bound to a 21-residue triple helical peptide, providing the only example to date of a vertebrate protein-collagen complex (10). More recent studies have identified a GVMGFO motif, conserved in collagens I-III...

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“…The mutations affected two residues that had previously been shown to directly interact with each other, forming an intramolecular salt bridge that is essential for SPARC binding to collagen type I. 26 Given the role of SPARC in collagen type I fiber assembly and the osteopenic phenotype of the SPARC-deficient mouse, 17,29 it is not unexpected that homozygous mutations in SPARC can lead to bone fragility in humans. Apart from severe bone fragility, individual 1 had severe early-onset scoliosis.…”

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confidence: 99%