Abstract: Expressed during many stages of development in a variety of organisms, the matricellular protein SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin or BM-40) is restricted in adult vertebrates primarily to tissues that undergo consistent turnover or to sites of injury and disease (1). The capacity of SPARC to bind to several resident proteins of the ECM, to modulate growth factor efficacy, to affect the expression of matrix metalloproteinases, and to alter cell shape as a counteradhesive factor, supports the idea that SPARC acts to regulate cell interaction with the extracellular milieu during development and in response to injury (Figure ​(Figure1;1; see also ref. 1). SPARC is a member of a gene family whose members share structural similarities in one or more protein domains (1). In addition to the numerous studies in cultured cells, the function of SPARC in vivo has been examined primarily in three evolutionarily diverse organisms — Caenorhabditis elegans, Xenopus laevis, and mice. These systems have been used to study the effects of increased or inappropriate SPARC expression, as well as diminished activity resulting from the inactivation of SPARC mRNA, the blocking of protein activity, or mutation of the SPARC gene (Table ​(Table1).1). This Perspective will integrate results from studies in vitro with findings in vivo in an attempt to clarify the current information and to propose functions for SPARC in living tissues.

Abstract: BACKGROUND Prostate cancer frequently metastasizes to bone. However, unlike many other tumors that produce osteolytic lesions, prostate cancer produces osteoblastic lesions through unknown mechanisms. In the current study, we explored the ability and mechanism of an osteotropic prostate cancer cell line (C4-2B) to induce mineralization. METHODS C4-2B cells were grown in promineralization media. Mineral deposition was characterized using von Kossa staining, calcium retention, alizarin red staining, Raman spectroscopy, and electron microscopy. Expression of osteoblast-related proteins was determined by RT-PCR. The nuclear level of the bone-specific transcription factor Cbfa1 was determined using western analysis and the effect of inhibiting Cbfa1 function, using a “decoy” Cbfa1 response element oligo, on mineralization was determined. RESULTS The studies demonstrated that C4-2B cells, but not its nonosteotropic parent cell line LNCaP, has an osteoblastlike phenotype including production of alkaline phosphatase, osteocalcin, osteonectin, bone sialoprotein, osteoprotegerin (OPG), and OPG ligand. Most importantly, the C4-2B cells produced hydroxyapatite mineral in vitro. Furthermore, C4-2B cells expressed high nuclear levels of the bone-specific transcription factor Cbfa1, compared to LNCaP cells, which accounts for their ability to produce bone-specific proteins. Inhibition of Cbfa1, using decoy DNA Cbfa1 response elements, abrogated the ability of C4-2B to produce mineral. Finally, we determined that C4-2B cells express bone morphogenic protein-7, a known inducer of Cbfa1 expression. CONCLUSIONS These data demonstrate a novel mechanism through which prostate cancer cells may directly contribute to the osteoblastic component that characterize their skeletal metastatic lesions. Prostate 47:212–221, 2001. © 2001 Wiley-Liss, Inc.

Abstract: We have identified secreted protein acidic and rich in cysteine (SPARC) as a potential glioma invasion-promoting gene. To determine whether SPARC alters the growth, attachment, or migration of gliomas, we have used U87T2 and doxycycline-regulatable SPARC-transfected clones to examine the effects of SPARC on (1) cell growth, (2) cell cycle progression, (3) cell attachment, and (4) cell migration, using growth curves, flow cytometry, attachment, and migration analyses on different brain ECMs, including collagen IV, laminin, fibronectin, vitronectin, hyaluronic acid, and tenascin. Our data indicate that SPARC delays tumor cell growth in the log phase of the growth curve. The clones secreted different levels of SPARC. The clone secreting the lowest level of SPARC was associated with a higher percentage of cells in G2M, whereas the clones secreting the higher levels of SPARC were associated with a greater percentage of cells in G0/G1. In comparison to the parental U87T2 clone, the SPARC-transfected clones demonstrated increased attachment to collagen, laminin, hyaluronic acid, and tenascin, but not to vitronectin or fibronectin. SPARC-transfected clones also demonstrated altered migration on the different extracellular matrix proteins. The modulation of migration, either positive or negative, was associated with changes in the level of secreted SPARC. These data suggest that SPARC may modulate glioma proliferation and invasion by modulating both the growth and migration of glioma cells.

Abstract: It is not known how gene expression of bone extracellular matrix molecules is controlled temporally and spatially, or how it is related with morphological differentiation of osteoblasts during embryonic osteogenesis in vivo. The present study was designed to examine gene expressions of type I collagen, osteonectin, bone sialoprotein, osteopontin, and osteocalcin during mandibular osteogenesis using in situ hybridization. Wistar rat embryos 13-20 days post coitum were used. The condensation of mesenchymal cells was formed in 14-day rat embryonic mandibles and expressed genes of pro-alpha 1 (I) collagen, osteonectin, bone sialoprotein and osteopontin. Cuboidal osteoblasts surrounding the uncalcified bone matrix were seen as early as in 15-day embryonic mandibles, while flat osteoblasts lining the surface of the calcified bone were seen from 16-day embryonic mandibles. Cuboidal osteoblasts expressed pro-alpha 1(I) collagen, osteonectin and bone sialoprotein intensely but osteopontin very weakly. In contrast, flat osteoblasts expressed osteopontin very strongly. Osteocytes expressed the extracellular matrix molecules actively, in particular, osteopontin. The present study demonstrated the distinct gene expression pattern of type I collagen, osteonectin, bone sialoprotein, osteopontin and osteocalcin during embryonic mandibular osteogenesis in vivo.

Abstract: The expression of SPARC (secreted protein acidic and rich in cysteine/osteonectin/BM-40) is elevated in endothelial cells participating in angiogenesis in vitro and in vivo. SPARC acts on endothelial cells to elicit changes in cell shape and to inhibit cell cycle progression. In addition, SPARC binds to and diminishes the mitotic activity of vascular endothelial growth factor. To determine the effect(s) of SPARC on angiogenic responses in vivo, we implanted polyvinyl alcohol sponges subcutaneously into wild-type and SPARC-null mice. On days 12 and 20 following implantation, SPARC-null mice showed increased cellular invasion of the sponges in comparison to wild-type mice. Areas of the sponge with the highest cell density exhibited the highest numbers of vascular profiles in both wild-type and SPARC-null animals. The endothelial component of the vessels was substantiated by immunoreactivity with three different markers specific for endothelial cells. Although sponges from SPARC-null relative to wild-type mice were populated by significantly more cells and blood vessels, an increase in the ratio of vascular to nonvascular cells was not apparent. No differences in the percentage of proliferating cells within the sponge were detected between wild-type and SPARC-null sections. However, elevated levels of vascular endothelial growth factor were associated with sponges from SPARC-null versus wild-type mice. An increase in vascular endothelial growth factor production was also observed in SPARC-null primary dermal fibroblasts relative to those of wild-type cells. In conclusion, we have shown that the fibrovascular invasion of polyvinyl alcohol sponges is enhanced in mice lacking SPARC, and we propose that increased levels of vascular endothelial growth factor account, at least in part, for this response.

Abstract: The OASIS gene, which encodes a novel CREB/ATF family member, was isolated from long-term cultured astrocytes that were employed as an in vitro gliosis model. In the present study, we examined the expression pattern of the OASIS gene in the developing mouse embryo by in situ hybridization histochemistry and compared it with the expression of osteogenesis markers. OASIS mRNA expression was most strongly detected in preosteoblasts of the outer bony cortex of the ribs. Alveolar bone also showed strong signals for OASIS gene expression. OASIS mRNA was also localized to the preodontoblast of tooth buds. Expression began at embryonic day 12 (D12.5), peaked around D14.5–16.5, and continued to D18.5. The pattern of expression was very similar to that of hXBP-1 mRNA, which encodes another CREB/ATF family member. Spatiotemporal patterns of OASIS partly overlapped that of osteopontin, osteonectin, and α1 type I procollagen genes. Among these, the time course of OASIS mRNA expression was most similar to that of osteopontin mRNA expression, suggesting that the OASIS protein is involved in the late phase of osteoblast differentiation, as compared to the Cbfa1 that regulates early phases of osteoblast differentiation.

Abstract: Alcohol is a risk factor for the development of osteoporosis, especially in men. Chronic alcohol abuse decreases bone mass, which contributes to the increased incidence of fractures. To better understand the mechanism of action of ethanol on bone metabolism, we have studied the dose-response effects of ethanol on conditionally immortalized human fetal osteoblasts (hFOB) in culture. Ethanol treatment had no significant effects on osteoblast number after 1 day or 7 days. Ethanol treatment did not reduce type I collagen protein levels at either time point at any dose but slightly reduced alkaline phosphatase activity after 7 days. The messenger RNA (mRNA) levels for alkaline phosphatase, type I collagen, and osteonectin were unaltered by 24 h of ethanol treatment but a high dose (200 mM) reduced mRNA levels for the two bone matrix proteins after 7 days. Ethanol treatment led to dose-dependent increases in transforming growth factor β1 (TGF-β1) mRNA levels and decreases in TGF-β2 mRNA levels. The concentration of ethanol in the medium decreased with time because of evaporation but there was little degradation caused by metabolism. These results, which show that cultured osteoblasts are less sensitive than osteoblasts in vivo, suggest that the pronounced inhibitory effects of ethanol on bone formation are not caused by direct cell toxicity.

Abstract: Human cells with osteogenic capacity were studied for differential gene expression. In the first part of the study we compared gene expression of marrow stroma cells (MSC) in comparison to matured osteoblasts cultured from trabecular bone (TBC) that were analyzed by RT-PCR for series of messages. High expression was detected for PTH-r, TGFb1 and biglycan in TBC compared to MSC's. The messages for c-MYC, IL-6, IL-11, M-CSF, osteonectin, and osteocalcin were expressed at the same level in the two populations of cells. In the second part of the study, we analyzed gene expression within the MSC derived from 25 donors (2.5–49 years old) with respect to donors' age and gender. Increased message levels for M-CSF and biglycan were measured in correlation with age of the donors. Gender differences did not affect the expression of cytokines studied (IL-6, IL-11, MCSF, TGFb1). We investigated the effect of Dexamethasone treatment on MSC and monitored an increased expression of IL-11, M-CSF, biglycan, and osteocalcin messages. This study employs primary cell systems (MSC and TBC) to illustrate differential gene expression by osteoblastic cells. The expression was correlated with maturation status of the cells with respect to differences between donors. J. Cell. Biochem. 83: 547–553, 2001. © 2001 Wiley-Liss, Inc.

Abstract: SPARC is a glycoprotein of the extracellular matrix that exhibits a number of biological functions such as disruption of cell adhesion and modulation of matrix metalloprotease expression. These properties, in concert with the expression of the molecule during development, repair, and neoplastic progression, suggest that SPARC has an important role in remodeling in a variety of tissues. However, the role of SPARC in the intestine is unclear since the development expression and tissular origin of SPARC in this organ appears to be species-dependent. As a first step to investigate the function of SPARC in the tissues of the intestine, we have analyzed its expression at the protein and mRNA levels in the human fetal and adult small intestinal and colonic mucosa as well as in intestinal cell models. Our results show that SPARC expression is differentially regulated during development and along the length of the human intestine. In the colon, SPARC was predominantly found at the epithelial-mesenchymal interface at the fetal stage, below detection levels in the normal adult, but re-expressed in the stroma of colonic tumors. In the small intestine, low levels of SPARC expression were observed at an early stage of morphogenesis (between 9 and 11 weeks) but expression was not detected at subsequent developmental stages nor was it induced in the mucosa of Crohn's disease. While SPARC appeared to be produced mainly by mesenchymal and stromal cells in the intact intestine it was not detected in colon cancer cells. Taken together, these results indicate that SPARC is subject to an onco-fetal pattern of expression in the stroma of the colonic mucosa while its expression is much more restricted in the small intestine, suggesting a differential involvement of this molecule in the extracellular matrix remodeling occurring along the length of the developing and diseased human intestinal mucosa.

Abstract: Background: Alcoholism is a risk factor for osteoporosis and it is not clear whether the detrimental effects of alcohol on bone are reversible. Parathyroid hormone (PTH) is a potent stimulator of bone matrix synthesis and is being investigated as a therapeutic agent to reverse bone loss. The present investigation was designed to determine the effects of PTH on bone formation in a rat model for chronic alcohol abuse. Methods and Results: Alcohol was administered in the diet of female rats (35% caloric intake) for 2 weeks. Human (1–34) PTH (80 μg/kg/day) was administered subcutaneously during the second week of the study. Alcohol resulted in a transient reduction in steady-state mRNA levels for the bone matrix proteins type 1 collagen, osteocalcin, and osteonectin compared with rats that were fed an alcohol-free (control) diet. As expected, alcohol decreased and PTH increased histologic indices of bone formation. Additionally, two-way ANOVA demonstrated that alcohol antagonized PTH-induced bone formation. Despite antagonism, bone formation and mRNA levels for bone matrix proteins in alcohol-fed rats treated with PTH greatly exceeded the values in rats fed the control diet. Conclusions: The results of this study contribute to a growing body of evidence that alcohol-induced bone loss is primarily due to reduced bone formation. We conclude that alcohol does not prevent the stimulatory effects of PTH on bone formation. This is evidence that the effects of alcohol on the skeleton are reversible. Additionally, the positive effects on bone formation in rats that consumed high concentrations of alcohol suggested that PTH may be useful as an intervention to treat alcohol-induced osteoporosis.

Abstract: Osteoblast differentiation under in vitro conditions is associated with increased expression of non-collagenous bone proteins including osteocalcin, osteopontin, and osteonectin, the exact function of which remain poorly understood To determine whether these proteins play an important role in the formation of mineralised bone matrix by osteoblasts in vivo, we analysed the time-course of their expression during estrogen-induced osteogenesis in female mice, and compared this with the formation of new cancellous bone Female mice were sacrificed prior to or following treatment with 17β-estradiol for up to 32 days (500 μg/animal/week) Total RNA was extracted from femurs, and changes in expression of genes for a range of osteoblast-derived proteins assessed by Northern blot analysis In parallel experiments, the time course of cancellous bone formation was determined by measuring bone mineral density (BMD) of the distal femur Estrogen led to a rapid increase in BMD, which reached significance by Day 16 This was preceded by three-fold increases in expression of alkaline phosphatase (ALP) and type I collagen (COL I) at Days 8 and 12 respectively In contrast, osteocalcin, osteopontin, and osteonectin expression showed no change during this initial period, although modest increases were observed at later times (ie, Days 20 and 24) Our results suggest that osteocalcin, osteopontin, and osteonectin are not involved in the initial phase of the osteogenic response to estrogen, suggesting that these non-collagenous bone proteins do not play a direct role in the formation of mineralised bone matrix by osteoblasts in vivo J Cell Biochem 82: 683–691, 2001 © 2001 Wiley-Liss, Inc

Abstract: Vitamin D deficiency elicits hypocalcified dentin. However, little is known about the action of vitamin D on the syntheses of dentin matrix proteins. In this study, we examined the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expressions of osteocalcin and osteonectin/secreted protein, acidic and rich in cysteine (SPARC), by human pulp cells in the presence or absence of transforming growth factor-β1 (TGF-β1) or basic fibroblast growth factor (bFGF). 1,25(OH)2D3 markedly increased osteocalcin at protein and mRNA levels. The osteocalcin level induced by 1,25(OH)2D3 was decreased and increased by TGF-β 1 and bFGF, respectively. 1,25(OH)2D3 suppressed SPARC synthesis at protein and mRNA levels. TGF-β1, but not bFGF, increased SPARC synthesis in the presence of 1,25(OH)2D 3. SPARC, but not osteocalcin, increased DNA synthesis in pulp cells. These findings suggest that 1,25(OH)2D3 and growth factors interactively regulate the expression of osteocalcin and SPARC in pulp cells, and that SPARC can sti...

Abstract: BACKGROUND Mechanical loading of bone is known to play a crucial role in bone remodeling and regeneration. Whereas the clinical effects of mechanically modulated bone healing have been extensively studied, less is known about the underlying mechanisms on a cellular level. This study was aimed at investigating the effects of uniaxial strains on osteoblast-like cells in culture. Mechanical loading was applied in physiological and hyperphysiological magnitudes. Nonstimulated cultures served as controls. RESULTS Cultured primary bovine periosteal cells exhibited phenotypic features of osteoblast-like cells. Application of physiological strains (2,000 mu strain) led to a bone-specific expression of extracellular matrix proteins (osteonectin, osteocalcin, collagen type I). Hyperphysiological loads (10,000 mu strain) were associated with an increased synthesis of proteoglycans. Proliferation of cells was higher than the controls at 10,000 mu strain and showed no difference from physiologically loaded osteoblasts. DISCUSSION Our study demonstrates that physiological loading of osteoblast-like cells enhances the regenerative capacity of bone, whereas hyperphysiological loads may impair bone regeneration.

Abstract: We have transduced adult human alveolar bone (AB) cells with a gene construct encoding a temperature-sensitive mutation of the SV40 large T antigen (tsT). Such cells divided rapidly, for more than 50 passages thus far, at a permissive low temperature (34.5°C), comparable to the non-transduced parental cells at 37°C. However, the tsT-transduced AB cells failed to grow at a non-permissive high temperature (39°C) at which the T antigen is inactivated. Nevertheless, the cells formed mineralised nodules in vitro at both the low and high temperatures. Flow cytometry analysis showed that the transduced cells cultured at 34.5°C, like the parental cells at 37°C, were smaller and less granular than the transduced cells incubated at 39°C. Moreover, the transduced cells grown at 34.5°C were also found to express bone sialoprotein, osteopontin and type I collagen at levels similar to those of the parental cells at 37°C, although osteonectin and fibronectin were down-regulated. When the transduced cells were incubated at 39°C, the expression of all antigens was up-regulated, particularly osteonectin. Thus, we have obtained long-term cultures of tsT-transduced AB cells whose growth is temperature-dependent and which express certain features characteristic of bone-derived cells.

Abstract: Tissue responses around implanted polymethylmethacrylate (PMMA) particles were analyzed by in situ hybridization with digoxigenin-labeled procollagen α1(I) (COL), osteonectin, osteocalcin, and osteopontin (OPN) mRNA probes. PMMA particles (150–300 μm in diameter) were implanted into rat tibiae, and specimens were collected at 3, 5, 7, and 10 days after operation. New bone was formed centripetally, and bone-forming osteoblasts expressed all four kinds of mRNAs. A COL signal was expressed most strongly and widely. In the early stage, COL-positive cells were detected on and among particles sporadically. A COL signal was rarely detected in cells on the surfaces of the particles, suggesting that PMMA particles may suppress osteoblast differentiation. Osteonectin and osteocalcin mRNAs were expressed in bone-forming osteoblasts in a similar pattern by day 7. By contrast, an OPN signal was detected mainly on the particles, not only in COL-positive osteoblasts but also in COL-negative round cells. The latter cells had acid phosphatase activity, suggesting that they might be macrophages responding to a foreign body. At day 10, an OPN signal was detected continuously in multinucleated cells on PMMA particles, whereas new bone was formed away from particles. Our approach helped us to understand the initial cellular reaction to materials, which may determine their biocompatibility. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res 54: 501–508, 2001

Abstract: Phyllodes tumor is a very rare neoplasm which accounts for 2.5% of all fibroepithelial lesions of the breast. The mesenchymal component of a malignant phyllodes tumor frequently contains heterologous components. We report a case of malignant phyllodes tumor. The patient was a 40-year-old woman with a lump on the left breast. Histological examination revealed the lump to be a malignant phyllodes tumor with foci of liposarcomatous differentiation. The mesenchymal tumor cells, including those in the liposarcomatous components, were found to express vimentin, osteonectin and vinculin. However, they showed no immunoreaction to CAM 5.2, desmin, alpha-smooth muscle actin (ASMA), neuron-specific enolase (NSE) nor S-100. Ultrastructurally, the mesenchymal tumor cells were found to have abundant cytoplasmic organelles, but there was no evidence showing their differentiation to myofibroblasts. Further studies will be necessary to elucidate the significance of vinculin and osteonectin expression in malignant phyllodes tumor.

Abstract: Fetal membranes overlying the cervix in patients prior to and during labour, and within the rupture tear after spontaneous delivery at term, exhibit altered morphology. In this study we report that in comparison to mid-zone fetal membranes biopsies, these regions are characterized by increased expression of the matricellular protein osteonectin or SPARC (Secreted Protein Acidic and Rich in Cysteine). In the reticular layer, the percentage of vimentin positive mesenchymal cells immunoreactive for osteonectin increased in these regions from 3–4% to 25– 33% and represented a fraction of the α-smooth muscle actin positive myofibroblasts elevated in the same regions. In the fibroblastic layer, the percentage of osteonectin positive cells increased from 1–5% to 8–13%; however, these did not exhibit the same relationship to the α-smooth muscle actin positive myofibroblasts in this layer. In the cytotrophoblastic layer the percentage of cytotrophoblastic cells immunoreactive for osteonectin increased from 1% to 6–12%. Elevation of in-situ detectable mRNA was also observed in the same cellular populations in this region. The incidence of cells positive for osteonectin mRNA or protein in the reticular layer correlated with morphological changes. Osteonectin has been implicated in the regulation of extracellular matrix turnover, and its pattern of expression suggests a role in the regional connective tissue and cytotrophoblastic changes proposed to be involved in the cleavage and rupture of fetal membranes.

Abstract: Several recent studies reflect major progress on the molecular biology and on the mechanisms of the lesions of fibrous dysplasia (monostotic and polyostotic forms) as well as the McCune-Albright syndrome. This review includes a detailed histological description of fibrous dysplasia: disorganized collagen fibers, woven bone formation, immature cytology of the osteoblasts and preosteoblastic cells. Histological lesions are also associated with an alteration of bone proteins: such as an increase in the expression of osteonectin, decrease of osteopontin and bone sialoprotein. Cell cultures of dysplasic osteoblastic cells have shown that the bone lesions are the result of abnormalities of the proliferation and differentiation of bone forming cells with a rise in CAMP levels. Poorly differentiated cells are associated with the formation of disorganized collagen fibers and with a trouble of synthesis of bone proteins. All these findings are linked to mutations of the Gs alpha (sub-unit alpha) protein. Various activating mutations are described as well as the molecular mechanisms of different forms of fibrous dysplasia, involving specially the c-fos proto-oncogene. Therapeutic implications are quoted, specially calcitonin and bisphosphonates.

Abstract: Osteoblast-like cells isolated from human bone bioptic specimens were established in culture. Their osteoblast-like phenotype was studied by biochemical, histochemical and immunohistochemical methods and by electron microscopy examination. Third-passage cell cultures exhibited high level of alkaline phosphatase activity and the exposure to human parathyroid hormone produced an increase of intracellular cAMP. Cultured cells were immunoreactive for type I and type III collagen, osteonectin, and fibronectin; when ascorbic acid and beta-glycerophosphate were added, they synthesized a rich extracellular matrix. This characterization ensures the reliability of osteoblast-like cultures when they are used as experimental models.

Abstract: +*Melrose, J (A-NHMRC); *Smith, S (A-NHMRC); **Little, C (A-The University of Wales); *Kitson, J (E-The Royal North Shore Hospital); *Hwa, S (A-Taiwanese Government); *Ghosh, P (E-University of Sydney) +*The Institute of Bone and Joint Research, The University of Sydney and Royal North Shore Hospital, St. Leonards, NSW 2065, Australia.. The Institute of Bone and Joint Research, Level 5, University Clinic, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia., 612-9926-7395, Fax: 612-9926-6539, jmelrose@mail.usyd.edu.au