Aurora Fausto

Erk is essential for growth, differentiation, integrin expression,and cell function in human osteoblastic cells

Extracellular signal-regulated kinases (Erks), members of the mitogen-activated protein kinase superfamily, play an important role in cell proliferation and differentiation. In this study we employed a dominant negative approach to determine the role of Erks in the regulation of human osteoblastic cell function. Human osteoblastic cells were transduced with a pseudotyped retrovirus encoding either a mutated Erk1 protein with a dominant negative action against both Erk1 and Erk2 (Erk1DN cells) or the LacZ protein (LacZ cells) as a control. Both basal and growth factor-stimulated MAPK activity and cell proliferation were inhibited in Erk1DN cells. Expression of Erk1DN protein suppressed both osteoblast differentiation and matrix mineralization by decreasing alkaline phosphatase activity and the deposition of bone matrix proteins. Cell adhesion to collagen, osteopontin, and vitronectin was decreased in Erk1DN cells as compared with LacZ cells. Cell spreading and migration on these matrices were also inhibited. In Erk1DN cells, expression of αβ1, αvβ3, and αvβ5 integrins on the surface was decreased. Metabolic labeling indicated that the synthesis of these integrins was inhibited in Erk1DN cells. These data suggest that Erks are not only essential for the growth and differentiation of osteoblasts but also are important for osteoblast adhesion, spreading, migration, and integrin expression.

Mechanically Strained Cells of the Osteoblast Lineage Organize Their Extracellular Matrix Through Unique Sites of αVβ3‐Integrin Expression

Bone cells transduce mechanical signals into anabolic biochemical responses. However, the mechanisms of mechanotransduction are unknown. To address this issue, we performed studies in primary cells of the human osteoblast lineage grown on collagen/vitronectin‐coated supports. We discovered that mechanical strain stimulated a redistribution of the αvβ3‐integrin to irregular plaque‐like areas at the cell‐extracellular matrix surface. Proteins involved in integrin‐matrix interactions in focal adhesions, vinculin and talin, did not localize to the plaque‐like areas of αvβ3‐expression, but signaling molecules such as focal adhesion kinase (FAK) did. Mechanical strain increased the number and size of the plaques defined by surface expression of αvβ3‐integrin. Osteopontin was secreted as a cross‐linked macromolecular complex, likely through the action of tissue transglutaminase that also was found in the plaques of αvβ3‐integrin cell‐matrix interaction. Mechanical strain increased mineralization of the extracellular matrix that developed in these plaques in αvβ3‐integrin‐dependent manner. Because the plaque‐like areas of cell‐matrix interaction exhibit macromolecular assembly and mineralization, we conclude that they may represent subcellular domains of bone formation and that αvβ3‐integrin activation represents one mechanism by which mechanical strain stimulates bone formation.

Regulation of ?V?3 and ?V?5 integrins by dexamethasone in normal human osteoblastic cells

Long‐term administration of pharmacological doses of glucocorticoids inhibits bone formation and results in osteoporosis. Since integrin‐mediated cell‐matrix interactions are essential for osteoblast function, we hypothesized that the detrimental effect of glucocorticoids on bone derived, at least in part, from decreased integrin‐matrix interactions. Because αvβ3 and αvβ5 integrins can interact with several bone matrix proteins, we analyzed the effects of dexamethasone (Dex) on the expression of these integrins in normal human osteoblastic cells. We found adhesion of these cells to osteopontin and vitronectin to be dependent on αvβ3 and αvβ5, respectively; this ligand specificity was not altered by Dex. The effects of Dex on the adhesion of human osteoblastic cells to osteopontin and vitronectin were biphasic with an increase after 2 days, followed by a decrease after 8 days of treatment. Consistently, surface αvβ3 and αvβ5 integrins, which were increased after 2 days of Dex treatment, were decreased after 8 days. Similarly, total cellular αv, β3, and β5 proteins, which were increased by Dex early in the culture, were diminished after 8 days. Metabolic labeling studies indicated that Dex exhibited biphasic regulation on the biosynthesis of αvβ5, with stimulation observed during the second day of treatment, followed by inhibition during the 8th day of exposure. By contrast, the biosynthesis of αvβ3 was inhibited by Dex on day 1 and remained inhibited on day 8. Analysis of the mRNA indicated that αv and β5 levels were increased by Dex during early exposure (1–3 days), followed by inhibition after prolonged exposure (≥7 days). By contrast, Dex decreased β3 mRNA level at all the time points analyzed. Consistently, Dex decreased β3 promoter activity after 1 day and persisted over 8‐day period. By contrast, Dex stimulated β5 promoter activity after 1 or 2 days but had no effect after 8 days. To further evaluate mechanism(s) leading to the decreased integrin expression after prolonged Dex treatment, mRNA stability was analyzed. Dex was found to accelerate the degradation of αv, β3 and β5 mRNA after an 8‐day treatment. Thus, the regulation of αvβ3 was dependent on transcription and posttranscriptional events whereas the expression of αvβ5 was dependent mainly on posttranscriptional events after prolonged Dex treatment. In conclusion, Dex exhibited time‐dependent regulation on the expression of αvβ3 and αvβ5 integrins in normal human osteoblastic cells. Short‐term exposure to Dex increased the levels of αvβ3 and αvβ5 on the surface and cell adhesion to osteopontin and vitronectin whereas long‐term exposure to Dex decreased the expression of both integrins and inhibited the cell adhesion to matrix proteins. J. Cell. Biochem. 77:265–276, 2000.

Regulation of insulin-like growth factors I and II and their binding proteins in human bone marrow stromal cells by dexamethasone

Glucocorticoids inhibit the proliferation, but induce the differentiation, of bone marrow stromal cells into osteoblast‐like cells. The mechanisms, however, are still conjectural. Since insulin‐like growth factors (IGFs) have profound effects on osteoblast growth and differentiation, it is possible that glucocorticoids exert their effects on bone marrow stromal cells in part via regulation of IGFs. Therefore, we analyzed the effects of dexamethasone (Dex) on the expression of IGF I and IGF II in cultured preosteoblastic normal human bone marrow stromal cells (HBMSC). Whereas Dex decreased the concentration of IGF I in the conditioned medium since early in the treatment, the concentration of IGF II was increased progressively as culture period lengthened. As the activities of IGF I and IGF II are regulated by the IGF binding proteins (IGFBPs), we analyzed the effects of Dex on the expression of IGFBPs. Dex increased IGFBP‐2 in a time‐dependent manner. The increase in IGFBP‐2, however, was only to the same extent as that of IGF II at most, depending on the length of treatment. Therefore, the increase in IGFBP‐2 would dampen, but not eliminate, the increased IGF II activities. By contrast, Dex decreased IGFBP‐3 levels, the latter increasing the bioavailability of IGF II. Although IGFBP‐4 mRNA levels were stimulated by Dex, IGFBP‐4 concentration in the conditioned medium was unchanged as measured by RIA. IGFBP‐5 and IGFBP‐6 mRNA levels were decreased by Dex in a time‐dependent fashion. IGFBP‐5 protein level was also decreased 1–4 days after Dex treatment. IGFBP‐1 mRNA was not detectable in HBMSC. These accumulated data indicate that Dex regulates IGF I and IGF II and their binding proteins differentially in normal human bone marrow stromal cells. The progressive increase in IGF II may contribute to Dex‐induced cell differentiation. J. Cell. Biochem. 71:449–458, 1998.

Bone deficit in ovariectomized rats. Functional contribution of the marrow stromal cell population and the effect of oral dihydrotachysterol treatment.

This study investigates the proliferative and osteogenic role of marrow stromal/osteoprogenitor cells in the development of the cortical bone deficit in ovariectomized (OVX) female rats. In vitro, clonal growth of marrow stromal cells from OVX rats was significantly impaired (vs. sham-operated controls). Yet in vivo, cells from sham-operated and OVX rats had equal osteogenic potential in several in vivo experimental situations, such as in intraperitoneally implanted millipore diffusion chambers and in intramuscular implants of marrow plus osteoinductive bone matrix (composite grafts). Long-term (6 mo) dihydrotachysterol (DHT) treatment of OVX rats enhanced their in vitro proliferative potential and clonal growth, as well as their osteogenic expression in composite grafts. The observation that the in vivo osteogenic performance of OVX rat marrow stromal cells was normal at extraosseous sites suggests that the mechanisms leading to osteopenia may involve an abnormality in cell-matrix interactions.

An Intact N Terminus Is Required for the Anabolic Action of Parathyroid Hormone on Adult Female Rats

Intermittent administration of parathyroid hormone (PTH) peptides increases bone density in animal and human models of osteoporosis. In vitro studies have demonstrated that PTH analogs lacking the first two amino acids can stimulate cell proliferation in certain cell systems, whereas fragments with an intact N terminus can be antimitogenic. We have tested whether the truncated PTH(3–38) fragment may be a better “anabolic analog” than PTH(1–38) by monitoring bone density and biomechanical properties of the femur in 6‐month‐old ovariectomized (OVX) rats. Either PTH fragment was administered subcutaneously (8 μg/100 g of body weight) 5 days/week, for 4 weeks, starting 1 week after surgery. During the entire study, untreated OVX rats lost 12.1 ± 4.4% of their initial bone density. PTH(1–38) reversed the initial bone loss, leading to complete restoration of presurgery values after 4 weeks of treatment. Conversely, administration of PTH(3–38) resulted in 13.2 ± 5.8% bone loss, while continuous estrogen infusion (10 μg/kg/day) prevented bone loss but did not reverse it. Sham‐operated animals also experienced significant bone loss in the vehicle and PTH(3–38)–treated groups (−4.5 ± 6.7%, and −7.6 ± 2.8%, respectively), whereas a significant gain in bone density (+4.4 ± 5.6%) was observed in the rats treated with PTH(1–38). A bone quality factor (index of strain energy loss) and the impact strength (resistance to fracture) were 25% and 44% lower in femurs explanted from OVX a0nimals treated with either vehicle or PTH(3–38), compared with sham‐operated animals. On the contrary, no difference was observed between OVX and control animals after treatment with PTH(1–38), indicating a preservation of the capacity to withstand mechanical stress. Thus, PTH(1–38) counteracts estrogen‐dependent loss of mineral density and bone biomechanical properties and increases bone density in estrogen‐replete animals. An intact N terminus sequence is necessary for this anabolic action of PTH.

Impaired Insulin Action in Rats with Non-insulin-dependent Diabetes

Studies were performed to test the hypothesis that impaired insulin action occurs as an acquired phenomenon in the streptozocin (STZ)-treated, non-insulin-dependent (NIDDM) diabetic rat model. A number of methods were used to evaluate impaired carbohydrate tolerance in these animals. Plasma glucose and insulin levels were measured at 6 and 14 wk of age, and insulin glucose tolerance tests were performed at 4 and 5 wk of age (before overt hyperglycemia ensues), and at 8 and 14 wk of age (after the animals manifest overt diabetes). The STZ-treated rats had higher plasma glucose levels than those of control animals (P < 0.001) at 6 and 14 wk of age, while their plasma insulin values were decreased to levels 73% of the controls (1.7 ng/ ml versus 2.3 ng/ml, P < 0.04). Glucose disappearance rates after high (0.35 U/kg) and low (0.175 U/kg) insulin challenge were reduced in the experimental diabetic animals at all ages with both insulin doses. The data suggest that these animals have an early and progressive acquired impairment in insulin action, most compatible with a defect in cellular biologic response to insulin. The possibility that this abnormality is secondary to insulin deficiency is raised.

Bone calcification and calcium homeostasis in rats with non-insulin-dependent diabetes induced by streptozocin

The effect of mild, non-insulin-dependent diabetes (NIDDM) on bone calcification and calcium (Ca) homeostasis was studied in growing rats (males and females). The diabetic state was characterized by mild insulin deficiency, plasma levels being 73% of controls, and mild hyperglycemia, with nonfasting plasma glucose levels of 1.5 times normal. There was no difference in plasma levels of Ca, phosphate (Pi), magnesium (Mg), alkaline phosphatase, immunoreactive parathyroid hormone (iPTH), calcitonin, 25-(OH)vitamin D (25[OH]D), 1,25-dihydroxyvitamin D (1,25[OHrsqb;2D), and 24,25-dihydroxyvitamin D (24,25[OH]2D) between the NIDDM rats and their controls of either sex. Metabolic Ca and Pi balance studies revealed that the experimental animals of both sexes were in positive Ca and Pi balance similar to that of their controls. Histologic studies of the kidney and intestinal slices from the experimental group were normal. Ca and Pi bone content calculated per gram bone ash of the femur, mandible, and second and fourth caudal vertebrae, and the organic content in the bones of the NIDDM animals showed no difference from their controls. Femur bone density and tibial epiphyseal growth plate width and morphology were similar histologically in the experimental and control rats. No decreased osteoid content in the tibial bone was found in the diabetic rats compared with controls. Physiologic sex differences, consisting of lower plasma Pi, higher plasma calcitonin levels, increased ratio of femur dry bone weight to total body weight, and increased percentage of mineralized and total bone volume at the tibial metaphysis seen in female compared with male control rats were also seen in the diabetic animals. The data reveal that, in states of mild insulin deficiency in the rat, the factors that regulate mineral metabolism are functioning normally and bone mineralization proceeds without alterations. In addition, physiologic sex differences are preserved. There is no indication for bone loss under these conditions.

25OHD, a circulating vitamin D metabolite in fish.

Abstract Serum and hepatic 25-hydroxyvitamin D (25OHD), and serum calcium, phosphate, 25OHD3 binding capacity and binding affinity were measured in male and female trout. Both serum and hepatic 25OHD levels are decreased in female trout with elevations in protein bound calcium and phosphate. Whereas the apparent dissociation constant (Kd) for serum binding of 25OHD3 of 1.0–2.0 × 10−9M is similar in males and females, the 25OHD3 binding capacity of hypercalcemic spawning trout (1.39 × 10−7M) is significantly less than that of male fish (1.88 × 10−7M). At circulating serum concentrations of 25OHD which average 9.5 × 10−9M only 5–7% of trout serum 25OHD binding sites are occupied.

Phenobarbital-induced Alterations in the Metabolism of [3H]Vitamin D3 by the Perfused Rachitic Rat Liver In Vitro

Anticonvulsant therapy of seizure disorders in man is associated with the development of complications involving bone and mineral metabolism including hypocalcemia, elevated serum immunoreactive parathyroid hormone levels, and increased amounts of unmineralized bone or osteoid. The latter has been attributed to a reduction in serum-25-hydroxycholecalciferol levels resulting from increased hepatic metabolism of vitamin D. Using an in vitro recycling hepatic perfusion system, we have demonstrated that 5 d of phenobarbital treatment increases the hepatic production of [(3)H]25-hydroxyvitamin D(3) (4.3+/-0.3 vs. 3.3+/-0.2%/h, P <0.025) without affecting the biliary excretion of radioactivity. Furthermore, rachitic livers perfused with blood obtained from animals treated with phenobarbital for 5 d also manifested an increase in [(3)H]25-hydroxyvitamin D(3) production (4.6+/-0.5 vs. 3.3+/-0.2%/h, P < 0.02). Addition of phenobarbital or its major metabolite, p-hydroxyphenobarbital, directly to the perfusion apparatus had no effect on [(3)H]25-hydroxyvitamin D(3) production. Phenobarbital treatment was also attended by a decrease in the intrahepatic content of [(3)H]vitamin D(3) (11.7+/-0.4 vs. 17.5+/-0.7 dpm/mg liver protein, P < 0.001) without alterations in the content of [(3)H]25-hydroxyvitamin D(3). The data collectively suggest that the increased hepatic conversion of [(3)H]vitamin D(3) to [(3)H]25-hydroxyvitamin D(3) attending phenobarbital treatment is secondary to stimulation of the hepatic 25-hydroxylation system(s) by a metabolite of phenobarbital other than p-hydroxyphenobarbital and/or by metabolic alterations resulting from phenobarbital therapy.