Human Secreted Protein Acidic And Rich In Cysteine Research Articles

Suppression of SPARC Ameliorates Ovalbumin-induced Airway Remodeling via TGFβ1/Smad2 in Chronic Asthma.

Airway remodeling is a critical feature of asthma. Secreted protein acidic and rich in cysteine (SPARC), which plays a cardinal role in regulating cell-matrix interactions, has been implicated in various fibrotic diseases. However, the effect of SPARC in asthma remains unknown. We studied the expression of SPARC in human bronchial epithelial cells and serum of asthmatics as well as in the lung tissues of chronic asthma mice. The role of SPARC was examined by using a Lentivirus-mediated SPARC knockdown method in the ovalbumin (OVA)-induced asthma mice. The biological processes regulated by SPARC were identified using RNA sequencing. The function of SPARC in the remodeling process induced by transforming growth factor β1 (TGFβ1) was conducted by using SPARC small interfering RNA (siRNA) or recombinant human SPARC protein in 16HBE cells. We observed that SPARC was up-regulated in human bronchial epithelia of asthmatics and the asthmatic mice. The levels of serum SPARC in asthmatics were also elevated and negatively correlated with the forced expiratory volume in one second (FEV1) to forced vital capacity ratio (FVC) (r = -0.485, P < 0.01) and FEV1 (%predicted) (r = -0.425, P = 0.001). In the chronic asthmatic mice, Lentivirus-mediated SPARC knockdown significantly decreased airway remodeling and airway hyper-responsiveness. According to gene set enrichment analysis, negatively enriched pathways found in the OVA + short hairpin-SPARC group included ECM organization and collagen formation. In the lung function studies, knockdown of SPARC by siRNA reduced the expression of remodeling-associated biomarkers, cell migration, and contraction by blocking the TGFβ1/Smad2 pathway. Addition of human recombinant SPARC protein promoted the TGFβ1-induced remodeling process, cell migration, and contraction in 16HBE cells via the TGFβ1/Smad2 pathway. Our studies provided evidence for the involvement of SPARC in the airway remodeling of asthma via the TGFβ1/Smad2 pathway.

Effect of SPARC Suppression in Mice, Perfused Human Anterior Segments, and Trabecular Meshwork Cells

PurposeSecreted protein, acidic and rich in cysteine (SPARC) elevates intraocular pressure (IOP), increases certain structural extracellular matrix (ECM) proteins in the juxtacanalicular trabecular meshwork (JCT), and decreases matrix metalloproteinase (MMP) protein levels in trabecular meshwork (TM) endothelial cells. We investigated SPARC as a potential target for lowering IOP. We hypothesized that suppressing SPARC will decrease IOP, decrease structural JCT ECM proteins, and alter the levels of MMPs and/or their inhibitors.MethodsA lentivirus containing short hairpin RNA of human SPARC suppressed SPARC in mouse eyes and perfused cadaveric human anterior segments with subsequent IOP measurements. Immunohistochemistry determined structural correlates. Human TM cell cultures were treated with SPARC suppressing lentivirus. Quantitative reverse transcriptase polymerase chain reaction (PCR), immunoblotting, and zymography determined total RNA, relative protein levels, and MMP enzymatic activity, respectively.ResultsSuppressing SPARC decreased IOP in mouse eyes and perfused human anterior segments by approximately 20%. Histologically, this correlated to a decrease in collagen I, IV, and VI in both the mouse TM and human JCT regions; in the mouse, fibronectin was also decreased but not in the human. In TM cells, collagen I and IV, fibronectin, MMP-2, and tissue inhibitor of MMP-1 were decreased. Messenger RNA of the aforementioned genes was not changed. Plasminogen activator inhibitor 1 (PAI-1) was upregulated in vitro by quantitative PCR and immunoblotting. MMP-1 activity was reduced in vitro by zymography.ConclusionsSuppressing SPARC decreased IOP in mice and perfused cadaveric human anterior segments corresponding to qualitative structural changes in the JCT ECM, which do not appear to be the result of transcription regulation.

SPARC enhances 5-FU chemosensitivity in gastric cancer by modulating epithelial-mesenchymal transition and apoptosis

Previous studies have shown that secreted protein acidic and rich in cysteine (SPARC) proteins can inhibit the development of cancer cells in various ways, such as by inhibiting angiogenesis and inhibiting cell proliferation. In fact, SPARC proteins may have an effect on the chemoresistance of gastric cancer cells to 5-Fluorouracil (5-FU), which needs further research in the future. Therefore, the purpose of this study was to explore the relationship between SPARC proteins and the chemosensitivity of gastric cancer cells to 5-FU. In vitro, after SPARC protein levels were regulated by plasmid, siRNA and human recombinant SPARC protein transfection in MGC-803, SGC-7901 and BGC-823 cells, we detected epithelial-mesenchymal transition (EMT), apoptosis markers and cell viability after 5-FU treatment. In vivo, we implanted BGC-823 cells with stable SPARC overexpression into nude mice. Tumour size was measured to assess the effect of SPARC protein on tumour formation and 5-FU chemosensitivity. In SGC-7901 and BGC-823 cells, both endogenous and exogenous upregulation of SPARC protein levels decreased cell viability, destroyed cytoskeletal F-actin, inhibited cell migration, and downregulated a series of transcription factors to inhibit cell EMT; it also upregulated cell apoptosis-related proteins to promote cell apoptosis. However, we obtained opposite results in SPARC knockdown MGC-803 cells. In vivo, compared with the control group, the group engrafted with BGC-823 cells stably overexpressing SPARC had a significant smaller tumour size. After 5-FU treatment, the new tumour gradually decreased in size. Our results show that the SPARC protein could enhance 5-FU chemosensitivity in gastric cancer cell lines by inhibiting EMT and promoting cell apoptosis.

Induction of SPARC on Oxidative Stress, Inflammatory Phenotype Transformation, and Apoptosis of Human Brain Smooth Muscle Cells Via TGF-β1-NOX4 Pathway.

Secreted protein acidic and rich in cysteine (SPARC) has a close association with inflammatory response and oxidative stress in tissues and is widely expressed in intracranial aneurysms (IAs), especially in smooth muscle cells. Therefore, it is inferred that SPARC might be involved in the formation and development of IAs through the inflammatory response pathway or oxidative stress pathway. The aim of this study is to investigate the pathological mechanism of SPARC in oxidative stress, inflammation, and apoptosis during the formation of IAs, as well as the involvement of TGF-β1 and NOX4 molecules. Human brain vascular smooth muscle cells (HBVSMCs) were selected as experimental objects. After the cells were stimulated by recombinant human SPARC protein in vitro, the ROS level in the cells was measured using an ID/ROS fluorescence analysis kit combined with fluorescence microscope and flow cytometry. The related protein expression in HBVSMCs was measured using western blotting. The mitochondrial membrane potential change was detected using a mitochondrial membrane potential kit and laser confocal microscope. The mechanism was explored by intervention with reactive oxygen scavengers N-acetylcysteine (NAC), TGF-β1 inhibitor (SD-208), and siRNA knockout. The results showed that SPARC upregulated the expression of NOX4 through the TGF-β1-dependent signaling pathway, leading to oxidative stress and pro-inflammatory matrix behavior and apoptosis in HBVSMCs. These findings demonstrated that SPARC may promote the progression of IAs.

Production and purification of recombinant human SPARC.

The matricellular protein SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin or as BM-40) is a collagen-binding protein with a capacity to induce cell rounding and influence proliferation in cultured cells. In mice that do not express SPARC, fibrillar collagen is reduced in some adult tissues; notably, a reduction in fibrosis is reported in response to fibrotic stimuli in lungs, heart, skin, liver, and in the eye. Recently, mutations in the gene encoding SPARC were found in patients afflicted with osteogenesis imperfecta. Thus, SPARC appears to be a critical mediator of collagen deposition and assembly in tissues. A useful tool for assessing the function of SPARC in ECM assembly is a source of purified recombinant SPARC. Outlined in this chapter is a brief discussion of different strategies for generating recombinant SPARC and an experimental strategy for producing and purifying human recombinant SPARC driven by baculoviral expression in insect cells.

A Mechanism Underlying Preventive Effect of High-Intensity Training on Colon Cancer.

We examined effects of high-intensity training on chemically induced aberrant crypt foci (ACF) in rat colon. We also investigated mechanisms that may underlie the results obtained, with a focus on secreted protein acidic and rich in cysteine (SPARC), which has been proposed as an exercise-related factor of colon cancer prevention. After an administration of 1,2-dimethylhydrazine, F344 rats executed high-intensity intermittent swimming training (HIIST) (twelve 20-s swimming with a weight [16% body weight] with 10-s pauses between the bouts) 5 d·wk for 4 wk. The acute and chronic effects of the HIIST on SPARC were evaluated in rats. We evaluated the in vitro and in vivo effects of 5' AMP-activated protein kinase (AMPK) activator on SPARC in rat serum and epitrochlearis muscle. In human subjects, we determined serum SPARC after exhaustive bicycling consisting of six to seven bouts of exercise at 170% V˙O2max with 10-s rests between the bouts (high-intensity intermittent bicycling [HIIB]). The SPARC mRNA in human vastus lateralis was measured before and after the HIIB for 4 d·wk for 6 wk (HIIB-training [HIIBT]). The numbers of ACF were lower in the HIIST (47 ± 22) compared with the control (122 ± 47) rats (P < 0.05). SPARC in epitrochlearis and serum after HIIS of the trained rat was higher than that in the control resting rats. In vitro and vivo AMPK stimulation increased mRNA and SPARC protein in rat epitrochlearis, respectively. The human serum SPARC after the HIIB was elevated. SPARC mRNA in human muscle was elevated after the HIIBT. The results demonstrated that HIIST inhibits 1,2-dimethylhydrazine-induced colon ACF development. This effect may be explained by SPARC induction by the exercise intensity-related factor AMPK, potentially explaining the preventive effects of high-intensity intermittent exercise training against colon cancer.

Bortezomib Attenuates Adhesion of B Cell Precursor Acute Lymphoblastic Lleukemia Cells to Bone Marrow Mesenchymal Stromal/Stem Cells Via Regulating SPARC Expression

The prognosis of adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is poor. Many cases in complete remission experience relapse of leukemia despite intensive chemotherapy or hematopoietic stem cell transplantation. This clinical observation suggests that minimal residual disease (MRD) still exists after these intensive therapies. Cell adhesion-mediated drug resistance (CAM-DR) is one of the mechanisms to support MRD in the bone marrow microenvironment (Clin Cancer Res 14:9, 2008). Mesenchymal stromal/stem cells (MSCs) are a cellular component of bone marrow (BM) and maintain physiological precursor B lymphopoiesis (Nature Rev Immunol 6:107, 2006). We investigated our hypothesis that survival of BCP-ALL cells is supported by their direct adhesion to BM-MSCs in BM microenvironment. First, we confirmed that BCP-ALL cells exhibited their drug resistant phenotype through adhesion to human BM-MSCs using human BCP-ALL cell line, Nalm6. We isolated human BM-MSCs from normal bone marrow samples (AllCells, Emeryville, CA). When Nalm6 cells were co-cultured with human BM-MSCs, they were prone to adhere to BM-MSCs. Nalm6 adhered to BM-MSCs (Nalm6/ad) were more resistant to the treatment with various anti-cancer drugs including doxorubicin than Nalm6 in suspension (Nalm6/su). Immunoblot analysis showed that expression level of anti-apoptotic protein Bcl-2 and phosphorylation level of pro-survival kinase Akt are higher in Nalm6/ad compared with those in Nalm6/su. In cell cycle analysis using Ki67/PI fluorescence-activated cell sorter (FACS) assay, the percentage of populations in G0 phase and S/G2/M phase was higher in Nalm6/ad compared with that in Nalm6/su, which indicated the increase of MRD and the high proliferation of leukemic cells in adhesive population, respectively. Therefore, we considered that detachment of Nalm6 from BM-MSCs is important to restore chemosensitivity of BCP-ALL cells and to eliminate these cells. Accordingly, we screened drugs that are capable of disrupting the adhesion of Nalm6 to BM-MSCs, and found that several proteasome inhibitors had a such activity. BM-MSCs were treated with bortezomib (10nM for 24h or 100nM for 1h), carfilzomib (3 nM for 24 h or 100 nM for 1 h) or oprozomib (10 nM for 24 h or 300 nM for 4 h), and then washed with PBS and co-cultured with Nalm6 cells. We confirmed that the number of BM-MSCs is not affected with these drugs irrespective of their concentration and incubation time by MTT assay. Co-cultured cells were divided into suspension cells and adherent cells. Each population was stained with monoclonal antibodies against CD19 and CD90 to detect Nalm6 and BM-MSCs, respectively, and analyzed by FACS. When BM-MSCs were pre-treated with bortezomib, carfilzomib or oprozomib, the number of Nalm6/ad was significantly decreased, and inversely, the number of Nalm6/su was increased, as compared to that of co-cultures with untreated BM-MSCs. Intriguingly, BM-MSCs treated with bortezomib showed aberrant expression of secreted protein acidic and rich in cysteine (SPARC). Immunoblot analysis showed that SPARC expression of BM-MSCs was transiently increased after bortezomib treatment. In co-cultures with BM-MSCs transfected with siRNA targeting SPARC, the number of Nalm6/ad was increased. In addition, the number of Nalm6 adhered to BM-MSCs pre-treated with recombinant human SPARC was lower than that adhered to control non-treated BM-MSCs. Therefore, SPARC showed anti-adhesive property and was one of the molecules associated with adhesion of BM-MSCs to Nalm6. Finally, we tested whether bortezomib shows therapeutic effects in vivo. Bortezomib administration supported survival of BCP-ALL xenograft model mice utilizing Nalm6, which was concomitant with the decrease in leukemia cells in the femur and in the size of spleen, as compared to those in non-treated control mice. In summary, Nalm6 cells that adhered to BM-MSCs contributed to construction of chemoresistant population similar to MRD. Here, we found that this population could be detached by treatment of BM-MSCs with bortezomib through transient increase in their SPARC expression. Our findings suggest that bortezomib or other proteasome inhibitors might be promising drugs to treat BCP-ALL through attenuating the adhesion of leukemic cells to BM-MSCs, and shed new insight into a therapeutic strategy in BCP-ALL by targeting BM-MSCs. Disclosures No relevant conflicts of interest to declare.

Integrative genomic analyses of secreted protein acidic and rich in cysteine and its role in cancer prediction.

Secreted protein acidic and rich in cysteine (SPARC), also termed osteonectin or basement‑membrane‑40 (BM‑40), is a matrix‑associated protein that elicits changes in cell shape, inhibits cell‑cycle progression and affects the synthesis of extracellular matrix (ECM). The final mature SPARC protein has 286 amino acids with three distinct domains, including an NH2‑terminal acidic domain (NT), follistatin‑like domain (FS) and C terminus domain (EC). The present study identified SPARC genes from 14 vertebrate genomes and revealed that SPARC existed in all types of vertebrates, including fish, amphibians, birds and mammals. In total, 21 single nucleotide polymorphisms (SNPs) causing missense mutations were identified, which may affect the formation of the truncated form of the SPARC protein. The human SPARC gene was found to be expressed in numerous tissues or organs, including in the bone marrow, whole blood, lymph node, thymus, brain, cerebellum, retina, heart, smooth muscle, skeletal muscle, spinal cord, intestine, colon, adipocyte, kidney, liver, pancreas, thyroid, salivary gland, skin, ovary, uterus, placenta, cervix and prostate. When searched in the PrognoScan database, the human SPARC gene was also found to be expressed in bladder, blood, breast, glioma, esophagus, colorectal, head and neck, ovarian, lung and skin cancer tissues. It was revealed that the association between the expression of SPARC and prognosis varied in different types of cancer, and even in the same cancer from different databases. It implied that the function of SPARC in these tumors may be multidimensional, functioning not just as a tumor suppressor or oncogene.

SPARC promotes the development of erythroid progenitors

Secreted protein acidic and rich in cysteine (SPARC) is a well-recognized regulator that affects cell proliferation, differentiation, and survival. Here, we investigated the impact of SPARC on erythropoiesis. Erythropoiesis in bone marrow (BM) from SPARC(-/-) mice was analyzed by flow cytometry and colony-forming assays. The mechanisms that affected erythropoiesis were investigated by BM transplantation experiments. CD34(+) cells from umbilical cord blood were isolated by MiniMACS, and the effect of SPARC on human erythropoiesis was assessed by colony-formation assay and erythroid differentiation culture. The hemoglobin level in peripheral blood (PB) was lower in SPARC(-/-) mice. Neither red blood cell count in PB nor the percentage of Ter119(+) erythrocytes in BM showed significant difference between SPARC(-/-) and WT mice. However, the ability of SPARC(-/-) bone marrow cells (BMCs) to form erythroid burst-forming units (BFU-E), as well as spleen colony-forming units (CFU-S8), was significantly decreased. The addition of exogenous SPARC could promote the formation of BFU-E from SPARC(-/-) BMCs in vitro. And the impaired ability of SPARC(-/-) BMCs to form BFU-E could be restored by transplanted into WT BM microenvironment, whereas the BFU-E formation of WT BMCs was impaired after transplanted into SPARC(-/-) recipients. Furthermore, exogenous SPARC promoted umbilical cord blood CD34(+) cells to form erythroid colonies and the hemoglobinization of erythroid, but did not affect the expression levels of glycophorin A and CD71. In conclusion, our results indicate that SPARC promotes the development of erythroid progenitors, but does not affect terminal erythroid differentiation.

SPARC modulates expression of extracellular matrix genes in human trabecular meshwork cells

To investigate the effects of secreted protein acidic and rich in cysteine (SPARC) on the expression of components of the extracellular matrix (ECM) in cultured human trabecular meshwork (TM) cells. Cultured human trabecular cells were transfected with small interfering RNAs (siRNAs) specific for the human SPARC gene. Protein and mRNA expressions of fibronectin (FN) and the α1chains of collagen I and collagen III were quantified. After silencing of the SPARC gene by transfection of cells with SPARC siRNA, the expression of COL1A1 and COL3A1 mRNAs and proteins was significantly enhanced, as compared to that in the control group (all, p < 0.001). In contrast, SPARC siRNA significantly reduced the expression of FN and SPARC mRNAs and FN protein, as compared to that in the control group (all, p < 0.001.). SPARC modulates the expression of several ECM genes in cultured human TM cells.

Effects of SPARC on expression of TGF-β1 and collagen type I in keloid fibroblasts

Objective To observe the effects of secreted protein, acidic and rich in cysteine （SPARC） on the expression of TGF-β1 and collagen type I in cultured human keloid fibroblasts by real-time fluorescence quantitative RT-PCR. Methods In vitro keloid fibroblasts were stimulated by different concentrations of recombinant human SPARC, and with the control group for comparison, real-time fluorescence quantitative RT-PCR to detect expression of TGF-β1 and collagen type I . Results Compared with the control group, the expression of TGF-β1 and collagen type I was significantly increased in the experimental group. Conclusions SPARC could enhance the expression of TGF-β1 and collagen type I in keloid fibroblasts significantly. Key words: Keloid;  Secreted protein, acidi and rich in cysteine;  quantitative reverse transcription-PCR;  Transforming growth factor-β1 ;  Real-time fluorescence Collagen type

SPARC Inhibits Adipogenesis by Its Enhancement of β-Catenin Signaling

SPARC (secreted protein acidic and rich in cysteine) modulates interactions between cells and extracellular matrix and is enriched in white adipose tissue. We have reported that SPARC-null mice accumulate significantly more fat than wild-type mice and maintain relatively high levels of serum leptin. We now show that SPARC inhibits adipogenesis in vitro. Specifically, recombinant SPARC inhibited (a) adipocyte differentiation of stromal-vascular cells isolated from murine white adipose tissue and (b) the expression of adipogenic transcription factors and adipocyte-specific genes. SPARC induced the accumulation and nuclear translocation of beta-catenin and subsequently enhanced the interaction of beta-catenin and T cell/lymphoid enhancer factor 1. The activity of integrin-linked kinase was required for the effect of SPARC on beta-catenin accumulation as well as extracellular matrix remodeling. During adipogenesis, fusiform preadipocytes change into sphere-shaped adipocytes and convert the extracellular matrix from a fibronectin-rich stroma to a laminin-rich basal lamina. SPARC retarded the morphological changes exhibited by preadipocytes during differentiation. In the presence of SPARC, the deposition of fibronectin was enhanced, and that of laminin was inhibited; in parallel, the expression of alpha5 integrin was enhanced, and that of alpha6 integrin was inhibited. Lithium chloride, which enhances the accumulation of beta-catenin, also inhibited the expression of alpha6 integrin. These findings demonstrate a role for SPARC in adipocyte morphogenesis and in signaling processes leading to terminal differentiation.

An In Vitro Model of Posterior Capsular Opacity: SPARC and TGF-β2 Minimize Epithelial-to-Mesenchymal Transition in Lens Epithelium

This report presents a novel model for studies of extracellular matrix (ECM) in posterior capsular opacification (PCO) in vitro. Lens epithelial cells (LEC) were cultured with an intraocular lens (IOL) on a surface of type IV collagen in an evaluation of the importance of the ECM-cell interaction in formation of PCO. Abnormal migration, proliferation, and expression of proteins associated with the epithelial-to-mesenchymal transition (EMT) that characterizes PCO were observed in the presence and absence of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine), which regulates matrix-cell interactions. The model for PCO in vitro consisted of an IOL placed on a membrane coated with collagen IV, a major constituent of the lens capsule. LECs from the lenses of wild-type (WT) and SPARC-null (SP-null) mice were cultured in the presence or absence of 10 ng/mL TGF-beta2 and 20 mug/mL recombinant human SPARC (rhSP) for up to 6 days. The migration of LECs was quantified. Labeling with BrdU and the measurement of DNA synthesis were assays for cell proliferation. Expression of the EMT markers, collagen type I, fibronectin, and alpha-smooth muscle actin were assessed using immunocytochemistry or Western immunoblots. LEC migration, proliferation, and the synthesis of EMT markers were enhanced in SP-null compared with WT LECs. TGF-beta2 inhibited the migration and proliferation of both WT and SP-null LECs in the presence of rhSP. TGF-beta2 increased the production of collagen type I, fibronectin, and alpha-SMA. The responses of SP-null LECs were rescued by the addition of recombinant human (rh)SP. A simple IOL culture system was useful for the evaluation of the effects of SPARC and TGF-beta2 on PCO in vitro. The action of TGF-beta2 on LEC migration and proliferation is influenced by SPARC, a regulator of matrix-cell interactions. The results indicate a functional intersection between pathways activated by TGF-beta2 and SPARC in the formation of PCO.

Porcine SPARC: isolation from dentin, cDNA sequence, and computer model

Genes encoding the major enamel matrix proteins and non-collagenous proteins of bone and dentin are members of the secretory calcium-binding phosphoprotein (SCPP) family, which originated from ancestral SPARC (secreted protein, acidic and rich in cysteine; BM-40/osteonectin). To better understand the role of SPARC in mineralizing systems, we isolated SPARC from developing pig teeth, deduced its primary structure from the cDNA sequence, and determined its quaternary structure by homology modelling with reference to human SPARC crystal structures. The guanidine/EDTA extract from porcine dentin was fractionated by anion-exchange and size-exclusion chromatography. Stains-all positive bands at 38 and 35 kDa gave the N-terminal sequences APQQEALPDETEV and DFEKNYNMYIFPV, which corresponded to the SPARC N terminus and an internal region of the protein. Porcine SPARC contains 300 amino acids, including the 17-amino acid signal peptide, and shares 96.2% amino acid sequence identity with human SPARC. Without post-translational modifications, the 283-amino acid secreted protein has a molecular mass of 32.3 kDa. The three-dimensional model revealed that porcine SPARC contains a single N-linked glycosylation at N113, seven intramolecular disulfide bridges, and assembles into dimers. SPARC is composed of three structural/functional domains: an acidic Ca2+-binding, a follistatin-like, and an extracellular calcium-binding domain.

Matricellular protein SPARC is translocated to the nuclei of immortalized murine lens epithelial cells

The matricellular glycoprotein, secreted protein acidic and rich in cysteine (SPARC), has complex biological activities and is important for lens epithelial cell function and regulation of cataract formation. To understand how SPARC influences lens epithelial cell activity and homeostasis, we have studied the subcellular distribution of SPARC in murine lens epithelial cells in vitro. We demonstrate that endogenous SPARC is located in the cytoplasm of either quiescent or dividing lens epithelial cells in culture. However, cytoplasmic SPARC was translocated into the nuclei of immortalized lens epithelial cells upon a significant reduction of intracellular SPARC in these cells. Recombinant human (rh) SPARC added to the culture media was quickly and efficiently internalized into the cytosol of SPARC-null lens epithelial cells. Moreover, cytoplasmic rhSPARC was also translocated into the nucleus after exogenous rhSPARC was removed from the culture media. The translocation of SPARC into the nucleus was therefore triggered by the reduction of SPARC protein normally available to the cells. A mouse SPARC-EGFP chimeric fusion protein (70 kDa) was expressed in lens epithelial cells and 293-EBNA cells, and was observed both in the cytoplasm and culture medium, but not in the nucleus. SPARC does not appear to have a strong nuclear localization sequence. Alternatively, SPARC might pass through the nuclear pore complex by passive diffusion. SPARC therefore functions not only as an extracellular protein but also potentially as an intracellular protein to influence cellular activities and homeostasis.

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.

Cu(ii) ion coordination to SPARC: a model study on short peptide fragments

SPARC (secreted protein, acidic and rich in cysteine) is a glycoprotein of the extracellular matrix that mediates the cell-matrix interactions. It plays also a role in angiogenesis, tumorigenesis, caractogenesis and wound healing. The human SPARC consists of three distinct modules. Module II is follistatin-like and its hydrolysis gives rise to a number of oligopeptides that can regulate angiogenesis in vivo and the biological activity of which has been related to their association with endogenous or exogenous copper ion. In order to completely understand the biological role of metal complexes formed by SPARC and its fragments, more information is needed on their stoichiometry, stability and structure in solution. In the present paper a potentiometric and spectroscopic investigation on Cu(II) complexes with the three SPARC122–126, SPARC121–126 and SPARC120–126 fragments, protected at both their amino and carboxylic ends, is reported. These peptides (Ac-HKLHL-NH2, Ac-GHKLHL-NH2 and Ac-KGHKLHL-NH2, respectively) constitute good models for the strong copper-binding site of the protein. The behaviour of the three ligands is very similar: complex formation is started by the two His residues, subsequently involving up to three amido nitrogens, as pH increases. The coordination of the two histydyl imidazoles promotes amide ionization in the physiological pH range and this can explain SPARC binding to the Cu(II) ion.

Cu(ii) ion coordination to the pentadecapeptide model of the SPARC copper-binding site

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular glycoprotein with many biological functions: it mediates the interactions between cells and the extracellular matrix, playing a role in angiogenesis, tumorigenesis, caractogenesis and wound healing. Proteolysis of SPARC gives rise to a number of oligopeptides which can regulate angiogenesis in vivo and the biological activity of which has been related to their association with endogenous or exogenous copper ion. Human SPARC consists of three distinct modules. Module II is follistatin-like and contains two copper binding sites, the strongest of which—the cationic region 2 (amino acids 114–130)—contains the sequence Gly–His–Lys. In order to shed more light on the biological role of metal complexes formed by SPARC and its fragments, more information is needed on their stoichiometry, stability and structure in solution. In the present paper a potentiometric and spectroscopic investigation on Cu(II) complexes with the SPARC114–128 fragment, protected at both its amino and carboxylic ends, is reported. This peptide (Ac–TLEGTKKGHKLHLDY–NH2) constitutes a good model to the strong copper-binding site of the protein. The whole experimental data suggest that complex-formation is started by the two His residues, subsequently involving up to three amido nitrogens, as pH increases. The coordination of the two histydyl imidazoles is able to promote amide ionisation in the physiological pH range and this could be the key to the SPARC affinity for Cu(II) ion.

Manipulating the aggregation and oxidation of human SPARC in the cytoplasm of Escherichia coli.

Human SPARC (secreted protein acidic and rich in cysteine), an extracellular matrix protein containing 14 cysteine residues, was found to partition equally between soluble and insoluble cellular fractions when overexpressed in the Escherichia coli cytoplasm. While the growth temperature and medium pH had little effect on inclusion body formation, co-overproduction of the dnaKJ operon, but not of the groE operon, suppressed aggregation at the expense of intracellular accumulation. Although both forms of the protein were fully reduced in wild-type cells, 70% to 85% of soluble and insoluble SPARC could be converted into oxidized species in a thioredoxin reductase (trxB) null mutant following incubation on ice. Approximately 15% to 20% of SPARC exhibited the electrophoretic mobility of the biologically active protein. Overproduction of the dnaKJ operon in trxB cells decreased the formation of disulfide-bonded SPARC multimers in the aggregated material but not in its soluble counterpart. Our results suggest that the activity responsible for disulfide bond formation in trxB mutants acts at the post-translational level and is able to freely diffuse within inclusion bodies.

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.