Ectopic Expression of SPARC inXenopusEmbryos Interferes with Tissue Morphogenesis: Identification of a Bioactive Sequence in the C-terminal EF Hand

Abstract

SUMMARY SPARC is a matricellular Ca 2 ϩ -binding glycoprotein that exhibits both counteradhesive and antiproliferative effects on cultured cells. It is secreted by cells of various tissues as a consequence of morphogenesis, response to injury, and cyclic renewal and/or repair. In an earlier study with Xenopus embryos we had shown a highly specific and regulated pattern of SPARC expression. We now show that ectopic expression of SPARC before its normal embryonic activation produces severe anomalies, some of which are consistent with the functions of SPARC proposed from studies in vitro. Microinjection of SPARC RNA, protein, and peptides into Xenopus embryos before endogenous embryonic expression generated different but overlapping phenotypes. (a) Injection of SPARC RNA into one cell of a two-cell embryo resulted in a range of unilateral defects. (b) Precocious exposure of embryos to SPARC by microinjection of protein into the blastocoel cavity was associated with certain axial defects comparable to those obtained with SPARC RNA. (c) SPARC peptides containing follistatin-like and copper-binding sequences were without obvious effect, whereas SPARC peptide 4.2, corresponding to a disulfide-bonded, Ca 2 ϩ -binding domain, was associated with a reduction in axial structures that led eventually to complete ventralization of the embryos. Histological analysis of ventralized embryos indicated that the morphogenetic events associated with gastrulation might have been inhibited. Microinjection of other Ca 2 ϩ -binding glycoproteins, such as osteopontin and bone sialoprotein, resulted in phenotypes that were unique. We probed further the structural correlates of this region of SPARC in the context of tissue development. Co-injection of peptide 4.2 with Ca 2 ϩ or EGTA, and injection of peptide 4.2K (containing a mutated consensus Ca 2 ϩ -binding sequence), demonstrated that the developmental defects associated with peptide 4.2 were independent of Ca 2 ϩ . However, the disulfide bridge in this region of SPARC was found to be critical, as injection of peptide 4.2AA, a mutant lacking the cystine, generated no axial defects. We have therefore shown for the first time in vivo that the temporally inappropriate presence of SPARC is associated with perturbations in tissue morphogenesis. Moreover, we have identified at least one bioactive region of SPARC as the C-terminal disulfide-bonded, Ca 2 ϩ -binding loop that was previously shown to be both counteradhesive and growth-inhibitory. (J Histochem Cytochem 45:643-655, 1997) E xtracellular matrix glycoproteins make major morphoregulatory contributions to early embryonic development. Their complex, transient expression patterns, structural heterogeneity, and functional diversity indicate important roles in the modulation of a broad spectrum of cellular activities, such as adhesion, migration, communication, and/or proliferation. For example, the blastocoel roof of amphibian embryos is coated with an elaborate network of fibronectin fibers before the involution of mesodermal cells durin...

Molecular evolution of SPARC: absence of the acidic module and expression in the endoderm of the starlet sea anemone, Nematostella vectensis

“…Our laboratory and others have demonstrated that a mimetic peptide corresponding to the unique C-terminal disulfide bridge of EF-hand2 of SPARC (peptide 4.2) has potent anti-proliferative and counteradhesive activities, both in vitro and in vivo (Damjanovski et al 1997;Lane and Sage 1990). While other mimetic peptides corresponding to other domains of SPARC have biological activity in vitro, to date only peptide 4.2 has been shown to mimic these effects in vivo.…”

Genome-wide expression analysis of therapy-resistant tumors reveals SPARC as a novel target for cancer therapy

“…Initial studies showed that SPARC was important in bone mineralization (18). Its role has been expanded to include tissue remodeling (19), endothelial cell migration (20,21), morphogenesis (19,22,23), and angiogenesis (24,25). SPARC has also been shown to have an antiproliferative effect on endothelial cells, mesangial cells, fibroblasts, and smooth muscle cells (26)(27)(28).…”

Calcium Affinity, Cooperativity, and Domain Interactions of Extracellular EF-hands Present in BM-40

“…Developmental anomalies are induced by overexpressing or suppressing BM-40 in nematodes (19,20). Furthermore, microinjection of BM-40 RNA, protein, peptides, or antibodies against BM-40 interfere with Xenopus embryonic development (21,22). In contrast, deletion of the BM-40 gene in mice resulted in a relatively mild phenoptype, namely late onset cataract formation in the eye, whereas development proceeded normally (23,24).…”