Louis V. Avioli

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.

Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein‐2

We have examined the effects of BMP‐2 on the expression of bone matrix proteins in both human bone marrow stromal cells (HBMSC) and human osteoblasts (HOB) and their proliferation and mineralization. Both HBMSC and HOB express BMP‐2/‐4 type I and type II receptors. Treatment of these two cell types with BMP‐2 for 4 weeks in the presence of β‐glycerophosphate and ascorbic acid results in mineralization of their matrix. BMP‐2 increases the mRNA level and activities of alkaline phosphatase and elevates the mRNA levels and protein synthesis of osteopontin, bone sialoprotein, osteocalcin, and α1(I) collagen in both cell types. Whereas the mRNA level of decorin is increased, the mRNA concentration of biglycan is not altered by BMP‐2. No effect on osteonectin is observed. The effect of BMP‐2 on bone matrix protein expression is dose dependent from 25 to 100 ng/ml and is evident after 1–7 days treatment. In the presence of BMP‐2, proliferation of HBMSC and HOB is decreased under either serum‐free condition or in the presence of serum. Thus, BMP‐2 has profound effects on the proliferation, expression of most of the bone matrix proteins and the mineralization of both relatively immature human bone marrow stromal preosteoblasts and mature human osteoblasts. J. Cell. Biochem. 67:386–398, 1997.

Effect of testosterone therapy on bone formation in an osteoporotic hypogonadal male

SummaryOsteoporosis has been reported to complicate androgen deficiency in males. Accordingly, we have evaluated an osteoporotic hypogonadal male with bone histomorphometry before and after 6 months of testosterone replacement. Androgen therapy resulted in increases in relative osteoid volum, total osteoid surface, linear extent of bone formation, and bone mineralization. The dramatic histological response to hormonal replacement confirms the importance of androgens in bone modeling and remodeling.

Effects of one-year treatment with estrogens on bone mass, intestinal calcium absorption, and 25-hydroxyvitamin D-1α-hydroxylase reserve in postmenopausal osteoporosis

SummaryA double-blind, placebo-controlled study on 21 postmenopausal osteoporotic women was performed in order to assess the effects of 1 year estrogen therapy (Premarin, 1.25 mg/day) on bone mass, intestinal calcium absorption, and mineral metabolism. Bone mineral content (BMC), measured by dual photon absorptiometry on the vertebral bodies and the femoral shaft, increased in both areas, but the changes were more evident at the former site, which is predominantly trabecular (+8.3%,P<0.05), than at the latter, which is mainly cortical (+2.6%,P<0.05). An improvement of intestinal calcium absorption was also detected at the end of the study (P<0.05) in the estrogen-treated group. Parameters of bone metabolism showed a decrease in hydroxyproline/creatinine ratio and osteocalcin, an increase in calcitonin, and no significant changes in parathyroid hormone (PTH) and alkaline phosphatase. Serum 1,25-dihydroxycholecalciferol (1,25(OH)2D3) levels increased after estrogen therapy, whereas 25-hydroxycholecalciferol (25OHD3) remained stable during the study period. Renal 25-hydroxyvitamin D 1α-hydroxylase reserve, assessed by the PTH-stimulation test, showed a more rapid response in producing a 1,25(OH)2D3 peak in the estrogen-treated patients compared with the control subjects. However, estrogens did not induce an absolute improvement in the secretory reserve. This study demonstrates that 1 year treatment with estrogens improves both intestinal calcium absorption and BMC in postmenopausal osteoporotic women. The latter effect appears to be induced by an inhibition of bone resorption, associated to an increased secretion of calcitonin, whereas vitamin D metabolites do not seem to contribute substantially to the mediation of estrogen action on bone.

In Vitro andIn Vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene

It has been well established that bone morphogenetic protein-2 (BMP-2) can induce bone formation bothin vivo andin vitro, although high concentrations (up to milligrams) of BMP-2 have been required to achieve this effectin vivo. Further, clinical applications are usually limited to a single dose at the time of implantation. In an attempt to prolong the transforming effect of BMP-2 we used a recombinant adenoviral vector carrying the human BMP-2 gene (Adv-BMP2) to transduce marrow-derived mesenchymal stem cells (MSC) of skeletally mature male New Zealand white rabbits. The pluripotential MSC were incubated with Adv-BMP2 overnight followed by culture in growth medium for 1 week. Assays on tissue cultures demonstrated that these Adv-BMP2 transduced MSC produced BMP-2 protein, differentiated into an osteoprogenitor line, and induced bone formationin vitro. These MSC had increased alkaline phosphatase activity, increased expression of type I collagen, osteopontin, and osteocalcin mRNA, and induced matrix mineralization compared with both nontransduced cells and cells transduced with a control adenoviral construct. To analyze the osteogenic potentialin vivo, Adv-BMP2-transduced MSC were autologously implanted into the intertransverse process space between L5 and L6 of the donor rabbits. The production of new bone was demonstrated by radiographic examination 4 weeks later in areas implanted with cells transduced with Adv-BMP2, whereas no bone was evident at sites implanted with cells transduced with the control adenoviral construct. Histological examination further confirmed the presence of new bone formation. These accumulated data indicate that it is possible to successfully transduce mesenchymal stem cells with a recombinant adenoviral vector carrying the gene for BMP-2 such that these cells will produce BMP-2, differentiate into an osteoprogenitor line, and induce bone formation bothin vitro andin vivo. Moreover, incubation of the Adv-BMP2-transduced cells for an additional 7 days in culture before transplantation enhances the success rate in bone formation (three out of three) as compared with our previous report (one out of five, Calcif Tissue Int 63:357–360, 1998).

Effects of prednisone and deflazacort on mineral metabolism and parathyroid hormone activity in humans.

SummaryThe effects of two different glucocorticoids, prednisone and deflazacort, (an oxazoline derivative of prednisolone) on bone metabolism were analyzed in 10 patients with disorders that required glucocorticoid therapy. Significant elevations in blood immunoreactive parathyroid hormone, alkaline phosphatase and urinary calcium, phosphate, hydroxyproline and nephrogenous cyclic AMP were observed during prednisone therapy in addition to an increase in the exchangeable calcium pool as estimated by47Ca-kinetic analyses. In contrast to these changes, deflazacort therapy induced minimal, and in some instances, no changes in these indices. In fact, in studies wherein prednisone therapy was followed by deflazacort alterations in bone metabolism, iPTH, and nephrogenous cAMP observed during prednisone were reversed. The data are consistent with the fact that the skeletal effects of prednisone therapy are mediated, at least in part, by increased parathyroid hormone activity, and that deflazacort is less potent in this regard.

Transforming Growth Factor-β Up-regulates the β5Integrin Subunit Expression via Sp1 and Smad Signaling

Integrin-mediated cell-matrix interactions play important roles in regulating cell function. Since transforming growth factor-β (TGF-β) modulates many osteoblast activities, we hypothesized that the growth factor acts in part by modulating integrin expression. TGF-β increased cell adhesion to vitronectin and up-regulated the surface level of αvβ5 via increasing β5 protein synthesis by a transcriptional mechanism. Promoter activity analysis demonstrated that a TGF-β-responsive element resides between nucleotides −63 and −44. Electrophoretic mobility shift assay and immunoprecipitation/Western studies indicated that the nuclear complex formed using the −66/−42 oligonucleotide contained both Sp1/Sp3 and Smad proteins. Since nuclear Sp1/Sp3 levels were not altered, whereas Smad levels were increased by TGF-β, we investigated the roles of Smad proteins in the up-regulation of β5 gene activation. Co-transfection of cells with β5 promoter reporter construct and expression vectors for Smad3, Smad4, and Sp1 increased the stimulatory effect of TGF-β. Furthermore, expression of dominant negative Smad3 or Smad4 in cells decreased or abolished the stimulation of β5 promoter activity by TGF-β. Smad4 mutant also inhibited the up-regulation of surface β5 level by TGF-β. Thus, TGF-β increases expression of the integrin β5 gene by mechanisms involving Sp1/Sp3 and Smad transcription factors.

Heparin-Induced Osteopenia: An Appraisal

Heparin has been used since 1936 for the treatment of vascular thrombotic episodes, but the association between heparin administration and osteopenia in man was not reported until 1964, when a causual notation was made regarding a spontaneous vertebral fracture in a 50-year-old man on heparin with no history of trauma (10). Jaffe and Willis (24) later reported the development of multiple symmetrical rib fractures, compression fractures of the thoracic vertebrae, and generalized skeletal demineralization in a 41-year-old man subjected to heparin at 20,000 units/day for 1 year. Earlier in the same year, Griffith et al. (18), reporting on 117 patients subjected to long-term heparin therapy, noted that 10 patients on subcutaneous heparin at 15,000 – 30,000 units/day for 6 months or longer developed spontaneous vertebral and/or rib fractures. These complications were not observed, however, in 107 patients receiving 10,000 units/day or less for 1 – 15 years. Bone biopsies obtained from two patients with osteopenia revealed that “the bony matrix was very soft, offering little or no resistance to the pathologist’s knife” (18).

Expression of bone matrix proteins during dexamethasone-induced mineralization of human bone marrow stromal cells.

Glucocorticoids have been shown to induce the differentiation of bone marrow stromal osteoprogenitor cells into osteoblasts and the mineralization of the matrix. Since the expression of bone matrix proteins is closely related to the differentiation status of osteoblasts and because matrix proteins may play important roles in the mineralization process, we investigated the effects of dexamethasone (Dex) on the expression of bone matrix proteins in cultured normal human bone marrow stromal cells (HBMSC). Treatment of HBMSC with Dex for 23 days resulted in a significant increase in alkaline phosphatase activity with maximum values attained on day 20 at which time the cell matrix was mineralized. Northern blot analysis revealed an increase in the steady‐state mRNA level of alkaline phosphatase over 4 weeks of Dex exposure period. The observed increase in the alkaline phosphatase mRNA was effective at a Dex concentration as low as 10−10 M with maximum values achieved at 10−8 M. In contrast, Dex decreased the steady‐state mRNA levels of both bone sialoprotein (BSP) and osteopontin (OPN) over a 4 week observation period when compared to the corresponding control values. The relative BSP and OPN mRNA levels among the Dex treated cultures, however, showed a steady increase after more than 1 week exposure. The expression of osteocalcin mRNA which was decreased after 1 day Dex exposure was undetectable 4 days later. Neither control nor Dex‐treated HBMSC secreted osteocalcin into the conditioned media in the absence of 1,25(OH)2D3 during a 25‐day observation period. The accumulated data indicate that Dex has profound and varied effects on the expression of matrix proteins produced by human bone marrow stromal cells. With the induced increment in alkaline phosphatase correlating with the mineralization effects of Dex, the observed concomitant decrease in osteopontin and bone sialoprotein mRNA levels and the associated decline of osteocalcin are consistent with the hypothesis that the regulation of the expression of these highly negatively charged proteins is essential in order to maximize the Dex‐induced mineralization process conditioned by normal human bone marrow stromal osteoprogenitor cells.

Bone Mineralization and Osteoblast Differentiation Are Negatively Modulated by Integrin αvβ3

Numerous bone matrix proteins can interact with αv‐containing integrins including αvβ3. To elucidate the net effects of the interaction between these proteins and αvβ3 on osteoblast function, we developed a murine osteoblastic cell line that overexpressed human αvβ3. Human αvβ3‐integrin was expressed on cell membrane, in which its presence did not alter the surface level of endogenous mouse αvβ3. The expressed human αvβ3 was functional because cell adhesion to osteopontin was increased and this increment was abolished by antibody against human αvβ3. The proliferation rate of cells overexpressing αvβ3 (αvβ3‐cells) was increased whereas matrix mineralization was decreased. To elucidate the mechanisms leading to inhibition of matrix mineralization, the expression of proteins important for mineralization was analyzed. Alkaline phosphatase activity and the expression of osteocalcin, type I collagen, and bone sialoprotein (BSP) were decreased whereas osteopontin was stimulated in αvβ3‐cells. The regulation of osteopontin, osteocalcin, and BSP expression was mediated via transcriptional mechanism because their promoter activities were altered. Examination of molecules involved in integrin signaling indicated that activator protein‐1 (AP‐1) and extracellular signal‐regulated kinase (Erk) activities were enhanced whereas c‐jun N‐terminal kinase (JNK) activity was decreased in αvβ3‐cells. The activity of p38 and the levels of focal adhesion kinase (FAK) and vinculin were not altered. Moreover, the adhesions of αvβ3‐cells to type I collagen and fibronectin were inhibited, which was attributed to decreased β1‐integrin levels on cell surface. In conclusion, overexpressing αvβ3‐integrin in osteoblasts stimulated cell proliferation but retarded differentiation, which were derived via altered integrin‐matrix interactions, signal transduction, and matrix protein expression.