Pharmacological regulation of SPARC by lovastatin in human trabecular meshwork cells.

PURPOSE: Statins have been shown to increase aqueous outflow facility. The matricellular protein SPARC (secreted protein acidic and rich in cysteine) is a critical mediator of aqueous outflow and intraocular pressure (IOP). Here, we examine the effects of lovastatin on SPARC expression in trabecular meshwork (TM) cells, exploring the molecular mechanisms involved. METHODS: Primary cultured human TM cells were incubated for 24, 48, and 72 hours with 10 µM lovastatin. In separate cultures, media was supplemented with either farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) for the duration of the 72-hour time point experiment. Trabecular meshwork cells were also pretreated for 24 hours with lovastatin followed by 24-hour stimulation with 3 ng/mL TGF-ß2. Cell lysates and media were harvested and relative mRNA and protein level changes were determined. Krüppel-like factor 4 (KLF4) localization in normal human anterior segments was examined by immunofluorescence. Adenovirus expressing human KLF4 was used and relative changes in SPARC mRNA and protein levels were assessed. RESULTS: Incubating TM cells with lovastatin suppressed SPARC mRNA and protein levels. This effect was reversed upon media supplementation with GGPP but not FPP. Pretreating cells with lovastatin inhibited TGF-ß2 induction of SPARC. The KLF4 transcription factor was expressed throughout the TM and the inner and outer walls of Schlemm's canal. Lovastatin treatment upregulated KLF4 mRNA and protein levels. Overexpression of KLF4 downregulated SPARC expression. CONCLUSIONS: Collectively, our data identify lovastatin as an important pharmacological suppressor of SPARC expression in TM cells, and provide further insight into the molecular mechanisms mediating statin enhancement of aqueous outflow facility.

Initial osteoblast-like cell response to pure titanium and zirconia/alumina ceramics.

OBJECTIVES: Outstanding mechanical properties, resistance to scratching and high biocompatibility make zirconia/alumina ceramics interesting for dental applications. To solve the problem of the well-known low temperature degradation and to provide stable mechanical properties a novel zirconia alloy ((Y,Nb)-TZP/alumina) was developed. The aim of this study was to investigate the initial bone cell response to this new zirconia/alumina composite ceramic. METHODS: HOS cells were cultured on zirconia/alumina composite (Zc) and pure titanium (Ti) discs. Surface topography was examined by atomic force microscopy (AFM), cell morphology by scanning electron microscopy (SEM). Cell proliferation (MTS) and alkaline phosphatase activity was measured at 1, 4 and 8 days. The mRNA expression of Cycline D1, the cell cycle regulating gene, integrin beta 1, osteonectin (ON) and beta-actin were evaluated by RT-PCR analysis after 12, 24 and 48 h. RESULTS: Both substrates showed a very smooth character with R(a)-values in the range of 0.002-0.113 microm supporting a continuous cellular growth. After 8 days, cell proliferation on Zc was higher than on Ti. The mRNA expression of cyclin D1 showed similar activity after 48 h on both surfaces, ALP activity was higher on Zc after 8 days. ON expression however showed no difference between the two groups. SIGNIFICANCE: Our data demonstrate that this new zirconia composite ceramic showed at least equivalent or slightly better biological response of osteoblast-like HOS cells than pure titanium during a short-time cell culture period.

Irradiation at 780 nm increases proliferation rate of osteoblasts independently of dexamethasone presence.

BACKGROUND AND OBJECTIVES: We have previously shown that phototherapy increases cell growth and impairs protein secretion of fibroblasts. Our objective was to study the effect of phototherapy on osteoblast-like cells in culture treated with dexamethasone. STUDY DESIGN/MATERIALS AND METHODS: Rat calvaria osteoblast-like cells were previously treated or not with dexamethasone and then, they were irradiated or not with a GaAlAs diode laser (wavelength of 780 nm, 10 mW, 3 J/cm2). Adhesion, proliferation, and osteonectin synthesis were analyzed. RESULTS: Phototherapy increased the proliferation rate of cells independently of dexamethasone presence. Adhesion and osteonectin synthesis were not significantly influenced by laser and/or dexamethasone. CONCLUSIONS: Based on the conditions of this study we concluded that phototherapy acts as a proliferative stimulus on osteoblast-like cells, even under the influence of dexamethasone. Thus, we suggest that phototherapy can be of importance as co-adjuvant in bone clinical manipulation in order to accelerate bone regeneration.

Characterization of mineral deposits formed in cultures of a hamster tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell line (CCP).

We have established tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell lines (CCP-2, CCP-7, CCP-8) from hamster bone marrow. Accumulation of mineral deposits was observed on the dishes when the clones were cultured in McCoy's 5A medium supplemented with 20% fetal calf serum. The materials were dissolved in 0.05 N HCl, and proteins found in the acid extracts were identified by N-terminal amino acid sequencing. The major components were bovine fetuin and prothrombin precursor. In addition, several cell-derived proteins, such as high mobility group 1 protein (HMG1), secretory leukocyte protease inhibitor (SLPI) and EPV20, a 2.0-kDa milk glycoprotein, were identified. HMG1 was detected, by immunostaining, on the cell surface of all the CCP clones. Metabolically labeled cellular sphingomyelin, sialyllactosylceramide, and proteoglycans were also found in the mineral deposits. Reverse transcription/polymerase chain reaction of CCP-2 mRNA revealed that the cells synthesized alkaline phosphatase, bone sialo protein, and osteonectin, but not matrix Gla protein, osteopontin, and type I collagen. CCP-2 cells formed tumors when injected subcutaneously into nude mice. In the tumor tissue, Alizarin-red-positive nodules surrounded by TRAP- and ALP-positive cells were observed, indicating CCP-2 cells can also induce calcification in vivo.

The effect of alkali- and heat-treated titanium and apatite-formed titanium on osteoblastic differentiation of bone marrow cells.

This study was based on the hypothesis that osteogenesis is enhanced by growth of osteogenic cells on an apatitic surface. To test this hypothesis, the behavior of rat bone marrow cells on these surfaces was examined: commercially pure titanium (Cp Ti), alkali- and heat-treated titanium (AH Ti), and AH Ti incubated in a simulated body fluid to deposit crystalline hydroxyapatite on the surface (Ap Ti). The alkaline phosphatase (ALP) activity of the cells cultured on Ap Ti was significantly higher at day 7 and day 14 than the ALP activity observed for the other titanium surfaces. At day 14, the ALP activity on AH Ti was significantly increased compared with the ALP activity on Cp Ti. The amount of DNA per well increased nearly in parallel for each titanium. However, northern blot analysis at day 14 revealed that expression of osteocalcin and alpha1(I) collagen mRNA was higher in the cells cultured on Ap Ti than the cells cultured on AH Ti. The cells cultured on Cp Ti showed the lowest mRNA levels. After 7 days of cell-free culture in medium supplemented with 15% serum, X-ray photoelectron spectroscopy (XPS), and thin-film X-ray diffraction (TF-XRD) analysis showed that calcium phosphate had been deposited on the AH Ti (resulting in an increase in thickness with time). No phosphate was detected on the Cp Ti, even after day 14. This study indicates that Ap Ti provides the most favorable conditions for differentiation of bone marrow cells, and, at a later stage, AH Ti also provides favorable conditions, perhaps because of the formation of a surface layer of calcium phosphate. This potential for apatite formation may play an important role in osteoblastic differentiation.

Effect of surface chemical modification of bioceramic on phenotype of human bone-derived cells.

In the search for methods to improve the biocompatibility of prosthetic materials, attention has recently been directed toward the potential use of surface chemical modification and its influence on cellular behavior. This in vitro study investigates the effect of surface chemistry modification of bioceramics on human bone-derived cells (HBDCs) grown on biomaterial surfaces for 2 weeks. Cells were cultured on either alumina (Al2O3), alumina doped with magnesium ions ([Mg]-Al2O3), or hydroxyapatite (HAP), as well as tissue culture polystyrene (TCPS). Expression of alkaline phosphatase (ALP), thrombospondin (Tsp), osteopontin (OP), osteocalcin (OC), osteonectin (ON/SPARC), type I collagen (Col I), and bone sialoprotein (BSP) were determined in terms of mRNAs and proteins. Protein levels for ALP, OP, OC, and BSP were significantly (p < 0. 05) greater at day 5 in HBDCs cultured on [Mg]-Al2O3 compared to those cells grown on Al2O3. At day 14 the levels of ALP, Tsp, Col I, OP, ON/SPARC, and BSP rose significantly (p < 0.05) above those occurring in HBDCs grown on Al2O3, HAP, and TCPS. This suggests that HBDCs from the same patient respond to differences in the surface chemical groups. This study confirms that the chemistry of a substratum, which facilitates cellular adhesion, will enhance cellular differentiation.

Impaired expression of noncollagenous bone matrix protein mRNAs during fracture healing in ascorbic acid-deficient rats.

In scorbutic patients, fractures are slow to heal because of impaired collagen synthesis. To investigate the influence of impaired collagen synthesis on the differentiation and proliferation of osteogenic and chondrogenic cells, we examined the expression of genes encoding bone matrix proteins, including osteonectin (ON), osteopontin (OPN), osteocalcin (OC), and matrix Gla protein (MGP), as differentiation markers for osteogenic and chondrogenic cells during fracture healing in Osteogenic Disorder Shionogi (ODS) rats, which have a hereditary defect in the ability to synthesize ascorbic acid (Asc). In ODS rats without Asc supplementation, intramembranous ossification was completely inhibited. Although a few fibroblast-like cells expressing ON mRNA were observed, no OPN mRNA-expressing cells were detected. During endochondral ossification, a small amount of metachromatic staining cartilage appeared at the fracture site, but there was no provisional calcification zone in the cartilage. Chondrocytes expressed ON and MGP mRNAs, but not OPN mRNA. When Asc was given to these rats, callus formation was soon detected around the fracture site, while OPN mRNA was expressed by differentiated osteoblasts and hypertrophic chondrocytes. Our data indicate that impaired collagen synthesis due to Asc deficiency inhibited the increase of ON and MGP mRNA-expressing cells as well as the appearance of OPN mRNA-expressing cells. Since OPN is considered to play an important role in normal and pathological mineralization, lack of OPN mRNA expression accompanying impaired collagen synthesis may have a role in defective mineralization and delayed fracture healing in scurvy.

Phase-Dependent effects of transforming growth factor beta 1 on osteoblastic markers of human osteoblastic cell line sV-HFO during mineralization.

A human osteoblastic cell line (SV-HFO) established in our laboratory expresses osteoblastic markers, including mineralization in vitro, in response to differentiation-inducing agents such as dexamethasone. In this study, we examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the mineralization of SV-HFO cells and show that TGF-beta 1 inhibited the mineralization of the cells via down regulation of tetranectin and alkaline phosphatase without influencing other osteoblastic markers. To examine precisely the effects of TGF-beta 1 on the process of mineralization, we tentatively divided the whole process of mineralization into four phases: induced ALP activity (days 0-5), maximal ALP activity (days 5-10), early mineralization (days 10-15), and progressive mineralization (days 15-20). These inhibitory effects of TGF-beta 1 on the expression of tetranectin and alkaline phosphatase, like that on mineralization, were observed only when TGF-beta 1 was applied in the early phase of the process of mineralization. On the other hand, the other osteoblastic markers were not influenced by treatment with TGF-beta 1. These results suggest that TGF-beta 1 may inhibit mineralization of osteoblasts by the downregulation of tetranectin and alkaline phosphatase expression in the early phase. Thus, TGF-beta 1 has phase-dependent effects on a human osteoblastic cell line during the process of mineralization.

Osteoblastic cells from rat long bone. I. Characterization of their differentiation in culture.

We here report the characterization for osteogenic markers alkaline phosphate (AP), osteocalcin, and mineral deposition of osteoblast cultures derived from cells migrating out from seven-day-old rat tibia fragments. The cells outgrown from bone fragments responded to stimulation with PTH with cAMP increase. We show in these cultures a high level of biosynthesis of type III collagen and osteonectin, and report the stimulatory effect that conditioned serum-free media (CM) from these cultures exert on the migration of endothelial cells (EA hy 926). The cultures were kept for the initial seven days in a Coon's modified F12 medium, then were switched to a medium containing ascorbic acid and beta-glycerophosphate (BGP), and cultured for another 41 days. They showed constitutive and timely restricted osteoblast markers and mineralization. Maximal AP activity occurred in concomitance with cell doubling, then fell to low levels by the time cultures were stationary. 45Ca incorporation in the monolayer increased after four weeks of culture, in concomitance with the appearance of unmineralized nodules, and remained high throughout the phase of mineral deposition. Biosynthesis of collagens type I, type III, and type V was detected at all times; secreted newly synthesized collagens decreased overall, relative to total secreted newly synthesized proteins, and on a per cell basis, with progression of the culture, while the ratio of collagen type III/collagen type I increased. Osteonectin was detected by immunohistochemistry and high amounts of osteonectin were synthesized constitutively. Osteocalcin was detected on virtually all cells tested at 21 and 28 days. A preliminary step in neoangiogenesis is the migration of endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in the expression of osteonectin, osteopontin and osteocalcin mRNAs during the development of skeletal tissues in vivo.

Heterogeneity in the expression of three members of non-collagenous matrix proteins in osteogenic and chondrogenic development in vivo were investigated by in situ hybridization. Sections of several skeletal tissues from mice at various stages of development were hybridized with digoxigenin-labeled complementary RNA probes encoding osteonectin (Osn), osteopontin (Osp) and osteocalcin (Osc). In calvariae and mandibulae, Osn messenger RNA (mRNA) was detected in cells in pre-osseous and osseous tissues before mineralization. Osp mRNA was found in cells attached to the mineralized bone matrix together with Osn mRNA followed by the expression Osc mRNA. In long bones, mRNAs for Osn, Osp and Osc were sequentially expressed with bone development from primary spongiosa to diaphyseal bone. In growth cartilage, Osn mRNA was observed in chondrocytes in non-mineralized cartilage, whereas Osp mRNA was detected in hypertrophic chondrocytes in mineralized cartilage matrix with a characteristic switch in expression. Osc mRNA was not detected in any chondrocytes. These results indicate that osteogenic differentiation in bone development in vivo is characterized by the sequential expression of these three genes, and suggest that these genes are expressed differentially and specifically, in association with extra-cellular matrix mineralization.

Expression of the gene encoding the extracellular matrix glycoprotein SPARC in the developing and adult mouse brain.

The pattern of expression of the SPARC gene was examined during postnatal development of the mouse brain using in situ hybridization. At postnatal day 3 (P3), a strong signal representing SPARC mRNA was apparent in boundary layers such as the pia mater and the lining of the ventricles. By P12, increased levels of SPARC mRNA were noted in the cerebellum, midbrain and brain stem with a lower signal in more frontal areas, a pattern which was retained in the adult. This pronounced caudal versus frontal difference in SPARC mRNA levels was confirmed by Northern blot analysis. At P3, SPARC mRNA was detected in developing blood vessels in the cerebral cortex, suggesting a role for SPARC in angiogenesis. During development of the cerebellum, expression of SPARC mRNA became highly restricted to the Purkinje cellular layer and in the adult was localized to Bergmann glial cells rather than Purkinje neurons.

The Caenorhabditis elegans homologue of the extracellular calcium binding protein SPARC/osteonectin affects nematode body morphology and mobility.

The extracellular matrix-associated protein, SPARC (osteonectin [Secreted Protein Acidic and Rich in Cysteine]), modulates cell adhesion and induces a change in cell morphology. SPARC expression in mammals is developmentally regulated and is highest at sites of extracellular matrix assembly and remodeling such as parietal endoderm and bone. We have isolated cDNA and genomic DNA clones encoding the Caenorhabditis elegans homologue of SPARC. The gene organization is highly conserved, and the proteins encoded by mouse, human, and nematode genes are about 38% identical. SPARC consists of four domains (I-IV) based on predicted secondary structure. Using bacterial fusion proteins containing nematode domain I or the domain IV EF-hand motif, we show that, like the mammalian proteins, both domains bind calcium. In transgenic nematodes expressing a SPARC-lacZ fusion gene, beta-galactosidase staining accumulated in a striated pattern in the more heavily stained muscle cells along the body. Comparison of the pattern of transgene expression to unc-54-lacZ animals demonstrated that SPARC is expressed by body wall and sex muscle cells. Appropriate levels of SPARC are essential for normal C. elegans development and muscle function. Transgenic nematodes overexpressing the wild-type SPARC gene were abnormal. Embryos were deformed, and adult hermaphrodites had vulval protrusions and an uncoordinated (Unc) phenotype with reduced mobility and paralysis.