James A. Bassuk

SPARC Regulates the Expression of Collagen Type I and Transforming Growth Factor-β1 in Mesangial Cells

The matricellular protein SPARC is expressed at high levels in cells that participate in tissue remodeling and is thought to regulate mesangial cell proliferation and extracellular matrix production in the kidney glomerulus in a rat model of glomerulonephritis (Pichler, R. H., Bassuk, J. A., Hugo, C., Reed, M. J., Eng, E., Gordon, K. L., Pippin, J., Alpers, C. E., Couser, W. G., Sage, E. H., and Johnson, R. J. (1997) Am. J. Pathol. 148, 1153–1167). A potential mechanism by which SPARC controls both cell cycle and matrix production has been attributed to its regulation of a pleiotropic growth factor. In this study we used primary mesangial cell cultures from wild-type mice and from mice with a targeted disruption of the SPARCgene. SPARC-null cells displayed diminished expression of collagen type I mRNA and protein, relative to wild-type cells, by the criteria of immunocytochemistry, immunoblotting, and the reverse transcription-polymerase chain reaction. The SPARC-null cells also showed significantly decreased steady-state levels of transforming growth factor-β1 (TGF-β1) mRNA and secreted TGF-β1 protein. Addition of recombinant SPARC to SPARC-null cells restored the expression of collagen type I mRNA to 70% and TGF-β1 mRNA to 100% of wild-type levels. We conclude that SPARC regulates the expression of collagen type I and TGF-β1 in kidney mesangial cells. Since increased mitosis and matrix deposition by mesangial cells are characteristics of glomerulopathies, we propose that SPARC is one of the factors that maintains the balance between cell proliferation and matrix production in the glomerulus.

Cleavage of the matricellular protein SPARC by matrix metalloproteinase 3 produces polypeptides that influence angiogenesis.

SPARC, a matricellular protein that affects cellular adhesion and proliferation, is produced in remodeling tissue and in pathologies involving fibrosis and angiogenesis. In this study we have asked whether peptides generated from cleavage of SPARC in the extracellular milieu can regulate angiogenesis. Matrix metalloproteinase (MMP)-3, but not MMP-1 or 9, showed significant activity toward SPARC. Limited digestion of recombinant human (rhu)SPARC with purified catalytic domain of rhuMMP-3 produced three major fragments, which were sequenced after purification by HPLC. Three synthetic peptides (Z-1, Z-2, and Z-3) representing motifs from each fragment were tested in distinct assays of angiogenesis. Peptide Z-1 (3.9 kDa, containing a Cu2+-binding sequence KHGK) exhibited a biphasic effect on [3H]thymidine incorporation by cultured endothelial cells and stimulated vascular growth in the chick chorioallantoic membrane (CAM). In contrast, peptides Z-2 (6.1 kDa, containing Ca2+-binding EF hand-1) and Z-3 (2.2 kDa, containing neither Cu2+-binding motifs nor EF hands), inhibited cell proliferation in a concentration-dependent manner and exhibited no effects on vessel growth in the CAM. Reciprocal results were obtained in a migration assay in native collagen gels: peptide Z-1 was ineffective over a range of concentrations, whereas Z-2 or Z-3 stimulated cell migration. Therefore, proteolysis of SPARC by MMP-3 produced peptides that regulate endothelial cell proliferation and/or migration in vitro in a mutually exclusive manner. One of these peptides containing KHGK also demonstrated a concentration-dependent effect on angiogenesis.

Human alpha B-crystallin - Small heat shock protein and molecular chaperone

The polymerase chain reaction was used to amplify a cDNA sequence encoding the human αB-crystallin. The amplified cDNA fragment was cloned into the bacterial expression vector pMAL-c2 and expressed as a soluble fusion protein coupled to maltose-binding protein (MBP). After maltose affinity chromatography and cleavage from MBP by Factor Xa, the recombinant human αB-crystallin was separated from MBP and Factor Xa by anion exchange chromatography. Recombinant αB-crystallin was characterized by SDS-polyacrylamide electrophoresis (PAGE), Western immunoblot analysis, Edman degradation, circular dichroism spectroscopy, and size exclusion chromatography. The purified crystallin migrated on SDS-PAGE to an apparent molecular weight (Mr ∼22,000) that corresponded to total native human α-crystallin and was recognized on Western immunoblots by antiserum raised against human αB-crystallin purified from lens homogenates. Chemical sequencing, circular dichroism spectroscopy, and size exclusion chromatography demonstrated that the recombinant crystallin had properties similar or identical to its native counterpart. Both recombinant αB-crystallin and MBP-αB fusion protein associated to form high molecular weight complexes that displayed chaperone-like function by inhibiting the aggregation of alcohol dehydrogenase at 37°C and demonstrated the importance of the C-terminal domain of αB-crystallin for chaperone-like activity.

Cell cycle-dependent nuclear location of the matricellular protein SPARC: association with the nuclear matrix.

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that inhibits cellular adhesion and proliferation. In this study, we report the detection of SPARC in the interphase nuclei of embryonic chicken cells in vivo. Differential partitioning of SPARC was also noted in the cytoplasm of these cells during discrete stages of M‐phase: cells in metaphase and anaphase exhibited strong cytoplasmic immunoreactivity, whereas cells in telophase were devoid of labeling. Immunocytochemical analysis of embryonic chicken cells in vitro likewise showed the presence of SPARC in the nucleus. Furthermore, elution of soluble proteins and DNA from these cells indicated that SPARC might be a component of the nuclear matrix. We subsequently examined cultured bovine aortic endothelial cells, which initially appeared to express SPARC only in the cytoplasm. However, after elution of soluble proteins and chromatin, we also detected SPARC in the nuclear matrix of these cells. Embryonic chicken cells incubated with recombinant SPARC were seen to take up the protein and to translocate it to the nucleus progressively over a period of 17 h. These observations provide new information about SPARC, generally recognized as a secreted glycoprotein that mediates interactions between cells and components of the extracellular matrix. The evidence presented in this study indicates that SPARC might subserve analogous functions in the nuclear matrix. J. Cell. Biochem. 74:152–167, 1999.

Fibroblast growth factor-10 is a mitogen for urothelial cells

Fibroblast growth factor (FGF)-10 plays an important role in regulating growth, differentiation, and repair of the urothelium. This process occurs through a paracrine cascade originating in the mesenchyme (lamina propria) and targeting the epithelium (urothelium). In situ hybridization analysis demonstrated that (i) fibroblasts of the human lamina propria were the cell type that synthesized FGF-10 RNA and (ii) the FGF-10 gene is located at the 5p12-p13 locus of chromosome 5. Recombinant (r) preparations of human FGF-10 were found to induce proliferation of human urothelial cells in vitro and of transitional epithelium of wild-type and FGF7-null mice in vivo.Mechanistic studies with human cells indicated two modes of FGF-10 action: (i) translocation of rFGF-10 into urothelial cell nuclei and (ii) a signaling cascade that begins with the heparin-dependent phosphorylation of tyrosine residues of surface transmembrane receptors. The normal urothelial phenotype, that of quiescence, is proposed to be typified by negligible levels of FGF-10. During proliferative phases, levels of FGF-10 rise at the urothelial cell surface and/or within urothelial cell nuclei. An understanding of how FGF-10 works in conjunction with these other processes will lead to better management of many diseases of the bladder and urinary tract.

REVIEW ARTICLE: THE MOLECULAR ERA OF BLADDER RESEARCH. TRANSGENIC MICE AS EXPERIMENTAL TOOLS IN THE STUDY OF OUTLET OBSTRUCTION

PURPOSE To review the crucial role of transgenic mice as experimental tools in the study of outlet obstruction. MATERIALS AND METHODS We reviewed the literature for studies that have used mice as models for outlet obstruction. RESULTS The combination of genetic manipulations and cellular physiology defines state-of-the-art experiments that explore the reciprocal mesenchymal-epithelial interactions that regulate bladder cell mechanisms. CONCLUSIONS The use of transgenic mice in bladder research has provided important data with respect to the molecular signals that drive bladder development, homeostasis, and the response to injury.

Spreading of embryologically distinct urothelial cells is inhibited by SPARC.

The AON epitope of secreted protein acidic and rich in cysteine (SPARC) is a conserved motif expressed by human SPARC in a variety of human cell types. Through the use of a monoclonal antibody that recognizes this epitope, transitional epithelium was found to restrict expression of SPARC to the suprabasal and intermediate layer. Such intracellular expression was defined by immunoreactive signals that localized to the apical plasma membranes of suprabasal and intermediate cells. Polarization of SPARC to apical plasma membranes of suprabasal cells was retained in vitro by a subpopulation of cells that exhibited characteristics of suprabasal cells—cell‐cycle quiescence, large cell volumes, and multiple nuclei. In contrast, the basal layer of transitional epithelium in vivo and cycling cells in vitro did not exhibit this apical staining pattern, but instead sequestered the SPARC polypeptide within urothelial cytoplasm and/or nuclei, as revealed by immunohistochemical analysis. Elution of soluble proteins and DNA from urothelial cells revealed the presence of SPARC within the nuclear matrix—and that SPARC colocalized with the nuclear matrix Ki‐67 antigen. rSPARC activity was demonstrated and quantified with a rounding assay whereby the spreading of freshly plated cells was inhibited by recombinant SPARC in a concentration‐ and time‐dependent manner. Inhibition of spreading was observed in urothelial cells derived from endoderm (bladder) and mesoderm (ureter) germ layers. Statistically significant differences were seen between urothelial cells from these two layers. Mesodermal cells recovered more slowly from the inhibitory effects of rSPARC, such that at hour 6 endodermal cells underwent significantly more spreading, as shown by a rounding index (RI). These experiments provide new insights about the matricellular trafficking of SPARC and suggest that intra‐ and extra‐cellular localization patterns influence the development, homeostasis, and differentiation of transitional epithelium.

The C-terminal Ca2+-binding domain of SPARC confers anti-spreading activity to human urothelial cells

The anti‐spreading activity of secreted protein acidic and rich in cysteine (SPARC) has been assigned to the C‐terminal third domain, a region rich in α‐helices. This “extracellular calcium‐binding” (EC) domain contains two EF‐hands that each coordinates one Ca2+ ion, forming a helix‐loop‐helix structure that not only drives the conformation of the protein but is also necessary for biological activity. Recombinant (r) EC, expressed in E. coli, was fused at the C‐terminus to a His hexamer and isolated under denaturing conditions by nickel‐chelate affinity chromatography. rEC‐His was renatured by procedures that simultaneously (i) removed denaturing conditions, (ii) catalyzed disulfide bond isomerization, and (iii) initiated Ca2+‐dependent refolding. Intrinsic tryptophan fluorescence and circular dichroism spectroscopies demonstrated that rEC‐His exhibited a Ca2+‐dependent conformation that was consistent with the known crystal structure. Spreading assays confirmed that rEC‐His was biologically active through its ability to inhibit the spreading of freshly plated human urothelial cells propagated from transitional epithelium. rEC‐His and rSPARC‐His exhibited highly similar anti‐spreading activities when measured as a function of concentration or time. In contrast to the wild‐type and EC recombinant proteins, rSPARC(E268F)‐His, a point substitution mutant at the Z position of EF‐hand 2, failed to exhibit both Ca2+‐dependent changes in α‐helical secondary structure and anti‐spreading activity. The collective data provide evidence that the motif of SPARC responsible for anti‐spreading activity was dependent on the coordination of Ca2+ by a Glu residue at the Z position of EF‐hand 2 and provide insights into how adhesive forces are balanced within the extracellular matrix of urothelial cells.

Renaturation of SPARC expressed in Escherichia coli requires isomerization of disulfide bonds for recovery of biological activity

SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin and BM-40) belongs to a group of secreted macromolecules that modulate cellular interactions with the extracellular matrix. During vertebrate embryogenesis, as well as in tissues undergoing remodeling and repair, the expression pattern of SPARC is consistent with a fundamental role for this protein in tissue morphogenesis and cellular differentiation. Human SPARC was cloned by the polymerase chain reaction from an endothelial cell cDNA library and was expressed in Escherichia coli as a biologically active protein. Two forms of recombinant SPARC (rSPARC) were recovered from BL21(DE3) cells after transformation with the plasmid pSPARCwt: a soluble, monomeric form that is biologically active (Bassuk et al., 1996, Archiv. Biochem. Biophys. 325, 8-19), and an insoluble form sequestered in inclusion bodies. Aggregated rSPARC was unfolded by urea treatment, purified by nickel-chelate affinity chromatography, and renatured by gradual removal of the denaturant. Proper isomerization of the disulfide bonds was achieved in the presence of a glutathione redox couple. After final purification by high resolution gel filtration chromatography, a monomeric form of rSPARC displaying biological activity was obtained. The recombinant protein inhibited the spreading and synthesis of DNA by endothelial cells, two properties characteristic of the native protein. We conclude that the information for the correct folding of rSPARC resides in the primary structure of the protein, and suggest that post-translational modifications are required neither for folding nor for biological activity.

Translocation of fibroblast growth factor-10 and its receptor into nuclei of human urothelial cells.

Fibroblast growth factor‐10 (FGF‐10), a mitogen for the epithelial cells lining the lower urinary tract, has been identified inside urothelial cells, despite its acknowledged role as an extracellular signaling ligand. Recombinant (r)FGF‐10 was determined by fluorescence microscopy optical sectioning to localize strongly to nuclei inside cultured urothelial cells. To clarify the possible role of a nuclear localization signal (NLS) in this translocation, a variant of rFGF‐10 was constructed which lacked this sequence. rFGF‐10(no NLS) was found in cytoplasm to a far greater degree than rFGF‐10, identifying this motif as a possible NLS. Furthermore, this variant displayed poor or non‐existent bioactivity compared to the wild‐type protein in triggering mitogenesis in quiescent urothelial cells. The presence of rFGF‐10(no NLS) in the nucleus suggested that additional interactions were also responsible for the nuclear accumulation of rFGF‐10. The FGF‐10 receptor was observed in cell nuclei regardless of the presence or concentration of exogenous rFGF‐10 ligand. Co‐localization studies between rFGF‐10 and the FGF‐10 receptor revealed a strong intracellular relationship between the two. This co‐localization was seen in nuclei for both rFGF‐10 and for rFGF‐10(no NLS), although the correlation was weaker for rFGF‐10(no NLS). These data show that an NLS‐like motif of rFGF‐10 is a partial determinant of its intracellular distribution and is necessary for its mitogenic activity. These advancements in the understanding of the activity of FGF‐10 present an opportunity to engineer the growth factor as a therapeutic agent for the healing of damaged urothelial tissue. J. Cell. Biochem. 102: 769–785, 2007.