Donna D. Strong

TWIST, a basic helix-loop-helix transcription factor, can regulate the human osteogenic lineage.

Basic helix‐loop‐helix (bHLH) transcription factors have been shown to play an important role in controlling cell type determination and differentiation. TWIST, a member of the bHLH transcription factor family, is involved in the development of mesodermally derived tissue, including the skeleton. We examined the role of human TWIST in osteoblast metabolism using stable expression of sense and antisense TWIST in human osteoblast HSaOS‐2 cells. Changes in morphology and osteogenic phenotype characterized these stable clones. Cells that overexpressed TWIST exhibited a spindle shaped morphology, reduced levels of alkaline phosphatase, a reduced proliferation rate, and failed to respond to basic fibroblast growth factor (bFGF). In contrast, those that underexpressed TWIST demonstrated a cuboidal epithelial‐like morphology characteristic of differentiated osteoblasts. TWIST antisense cells exhibited increased levels of alkaline phosphatase and type I collagen mRNA, initiated osteopontin mRNA expression, and had a reduced proliferation rate. These results indicate that TWIST overexpressing cells may de‐differentiate and remain in an osteoprogenitor‐like state, and antisense TWIST cells progress to a more differentiated mature osteoblast‐like state. Therefore, the level of TWIST can influence osteogenic gene expression and may act as a master switch in initiating bone cell differentiation by regulating the osteogenic cell lineage. J. Cell. Biochem. 75:566–577, 1999.

Structure-function analysis of the human insulin-like growth factor binding protein-4.

To identify the molecular mechanism by which insulin-like growth factor binding protein-4 (IGFBP-4) exerts its inhibitory effects on insulin-like growth factor (IGF) actions, we localized and determined the role of the IGF binding domain in modulating IGF actions in human osteoblasts. Deletion analysis using IGFBP-4 expressed in bacteria revealed that the N-terminal sequence Leu72–Ser91 was essential for IGF binding. The C-terminal fragments (His121–Glu237 or Arg142–Glu237) did not bind to IGF but loss of these regions decreased IGF binding activity. Detailed deletion analysis identified the residues Cys205–Val214as the motif to facilitate IGF binding. Mitogenic studies revealed that an IGFBP-4 mutant (His74 replaced by Pro74) and an N-terminal peptide (N terminus to Thr71) with little IGF binding activity failed to inhibit IGF-II-induced human osteoblast proliferation. An N-terminal peptide (N terminus to Asn182) with reduced IGF binding activity inhibited IGF action but with lower potency. In contrast, an IGFBP-4 mutant (His74 replaced with Ala74) exhibited similar IGF binding activity and potency in inhibiting the activity of IGF-II compared with the wild type. Therefore, the N-terminal sequence (Leu72–Ser91) and the C-terminal sequence (Cys205–Val214) are necessary to form the high affinity IGF binding domain, which is the major structural determinant of the IGFBP-4 function.

Insulin-like growth factor-I and insulin-like growth factor-II induce c-fos in mouse osteoblastic cells

SummaryWe investigated the expression of c-fos in mouse osteoblast-like cultures treated with insulin-like growth factor (IGF)-I and IGF-II. The IGFs are present in bone, are produced by osteoblast-like cells in culture, and stimulate osteoblast cell proliferation. Quiescent, subconfluent cultures of the clonal osteoblast-like mouse calvarial cell line, MC3T3-E1, were treated with 10 ng/ml of IGF-I or IGF-II. RNA was extracted at 0, 15, 30, 60, 120 and 240 minutes, and c-fos messenger RNA (mRNA) was analyzed on Northern blots. Both IGFs transiently increased c-fos mRNA levels 25–28 fold at 15–30 min. To determine if c-fos induction was unique to the MC3T3-E1 cell line, effects of IGF-1 and IGF-II (3 ng/ml) were also tested in quiescent, serum-free primary mouse calvarial cells. Levels of c-fos mRNA were increased at 15 and 30 minutes (40-fold with IGF-I and 5-fold with IGF-II). These results indicate that IGF-I and IGF-II caused a rapid and transient induction of c-fos mRNA in murine osteoblasts.

Differentiation of normal human bone cells by transforming growth factor-β and 1,25(OH)2 vitamin D3

To investigate the role of transforming growth factor-beta 1 (TGF beta) in bone metabolism, the effects of this agent on the differentiation characteristics of human bone cells were studied in vitro. Human bone cells were isolated from femoral head samples by collagenase digestion. Differentiation characteristics included alkaline phosphatase activity, osteocalcin production, and mRNA levels for alkaline phosphatase, type I alpha 2-procollagen, and osteocalcin. The effect of TGF beta on alkaline phosphatase was not constant, but varied with the incubation conditions. At high cell density and in the presence of serum, TGF beta decreased alkaline phosphatase activity. However, at low cell density and under serum-free conditions, TGF beta stimulated alkaline phosphatase activity. The addition of 1,25(OH)2 vitamin D3 also stimulated alkaline phosphatase. The combination of the two agents gave a greater increase in activity than the sum of the activities when the two agents were given alone. The percentage of cells that stain positively for alkaline phosphatase changed in parallel with the change in specific activity. The percentage of positive cells increased from 17% to 64%, while the specific activity increased from 22 to 169 mU/mg protein. To investigate the mechanism of this stimulation, mRNA levels were measured at 24 hours. Individually, TGF beta and 1,25(OH)2D3 increased message levels for alkaline phosphatase and type I procollagen, but the greatest effect was produced by the combination of the two factors. 1,25(OH)2D3 increased osteocalcin mRNA levels, but TGF beta markedly inhibited this stimulation. TGF beta also inhibited production of osteocalcin by the human bone cells. TGF beta appears to modulate differentiation of human bone cells in combination with 1,25(OH)2D3 and other factors.

Studies on the Role of Human Insulin‐like Growth Factor‐II (IGF‐II)‐Dependent IGF Binding Protein (hIGFBP)‐4 Protease in Human Osteoblasts Using Protease‐Resistant IGFBP‐4 Analogs

To characterize the insulin‐like growth factor binding protein‐4 (IGFBP‐4) protease produced by human osteoblasts (hOBs), we localized and determined the role of the proteolytic domains in human IGFBP‐4 (hIGFBP‐4) in modulating IGF‐II actions. N‐terminal amino acid sequence and mass spectrometric analyses of the 6xHis‐tagged IGFBP‐4 proteolytic fragments revealed that Met135‐Lys136 was the only cleavage site recognized by the IGF‐II–dependent IGFBP‐4 protease produced by hOBs. This cleavage site was confirmed by the finding that deletion of His121 to Pro141 blocked proteolysis. However, unexpectedly, deletion of Pro94 to Gln119 containing no cleavage site had no effect on IGF‐II binding activity but blocked proteolysis. Addition of the synthetic peptide corresponding to this region at concentrations of 250 or 1000 molar excess failed to block IGFBP‐4 proteolysis. These data suggest that residues 94–119 may be involved in maintaining the IGFBP‐4 conformation required to expose the cleavage site rather than being involved in direct protease‐substrate binding. To determine the physiological significance of the IGF‐II–dependent IGFBP‐4 protease, we compared the effect of the wild‐type IGFBP‐4 and the protease‐resistant IGFBP‐4 analogs in blocking IGF‐II–induced cell proliferation in normal hOBs, which produce IGFBP‐4 protease, and MG63 cells, which do not produce IGFBP‐4 protease. It was found that protease‐resistant IGFBP‐4 analogs were more potent than the wild‐type protein in inhibiting IGF‐II–induced cell proliferation in hOBs but not in MG63 cells. These data suggest that IGFBP‐4 proteolytic fragments are not biologically active and that IGFBP‐4 protease plays an important role in regulating IGFBP‐4 bioavailability and consequently the mitogenic activity of IGFs in hOBs.

Retroviral‐based gene therapy with cyclooxygenase‐2 promotes the union of bony callus tissues and accelerates fracture healing in the rat

An in vivo gene therapy strategy was developed to accelerate bone fracture repair.

Osteogenic protein-1 stimulates mRNA levels of BMP-6 and decreases mRNA levels of BMP-2 and -4 in human osteosarcoma cells.

Bone morphogenetic proteins (BMPs) are novel growth and differentiation factors that act on mesenchymal stem cells to initiate new bone formation in vivo and promote the growth and differentiation of cells in the osteoblastic lineage. In the present study, we examined the effects of recombinant human osteogenic protein-1 (also known as BMP-7) on the expression of related members of the BMP family using SaOS-2 and U2-OS, two human osteosarcoma cell strains. Evaluation of BMP-2, -4, and -6 mRNA expression indicates that OP-1 stimulated the mRNA levels of BMP-6 in both SaOS-2 cells (threefold) and U2-OS cells (fivefold) after 24 hours of treatment, while decreasing the mRNA levels of BMP-4 in SaOS-2 cells (80%) and BMP-2 and BMP-4 in U2-OS cells by 50% and 72%, respectively. BMP-2 mRNA expression, as examined by Northern blot analysis, was below detectable limits in SaOS-2 cultures. These results demonstrate that OP-1 modulates the mRNA expression of related members of the BMP family, suggesting a possible mode of action of OP-1 on the growth and differentiation of cells in the osteoblastic lineage in vitro.

Nuclear factor I transcription factors regulate IGF binding protein 5 gene transcription in human osteoblasts.

Insulin-like growth factor binding protein 5 (IGFBP5) is expressed in many cell types including osteoblasts and modulates IGF activities. IGFBP5 may affect osteoblasts and bone formation, in part by mechanisms independent of binding IGFs. The highly conserved IGFBP5 proximal promoter within 100 nucleotides of the start of transcription contains functional cis regulatory elements for C/EBP, Myb and AP-2. We report evidence for a functional Nuclear Factor I (NFI) cis element that mediates activation or repression of IGFBP5 transcription by the NFI gene family. All four NFI genes were expressed in human osteoblast cultures and osteosarcoma cell lines. Co-transfection with human IGFBP5 promoter luciferase reporter and murine Nfi expression vectors showed that Nfib was the most active in stimulating transcription. Nfix was less active and Nfia and Nfic were inhibitory. Knockdown of NFIB and NFIC expression using siRNA decreased and increased IGFBP5 expression, respectively. Analysis of IGFBP5 promoter deletion and mutation reporter constructs identified a functional NFI cis element. All four NFI proteins bound the NFI site in electrophoretic mobility shift experiments and NFIB bound in chromatin immunoprecipitation assays. Results suggest that NFI proteins are important regulators of IGFBP5 expression in human osteoblasts and thus in modulating IGFBP5 functions in bone.

Potential involvement of the interaction between insulin-like growth factor binding protein (IGFBP)-6 and LIM mineralization protein (LMP)-1 in regulating osteoblast differentiation.

Insulin‐like growth factor binding protein (IGFBP)‐6 has been reported to inhibit differentiation of myoblasts and osteoblasts. In the current study, we explored the mechanisms underlying IGFBP‐6 effects on osteoblast differentiation. During MC3T3‐E1 osteoblast differentiation, we found that IGFBP‐6 protein was down‐regulated. Overexpression of IGFBP‐6 in MC3T3‐E1 and human bone cells inhibited nodule formation, osteocalcin mRNA expression and ALP activity. Furthermore, accumulation of IGFBP‐6 in the culture media was not required for any of these effects suggesting that IGFBP‐6 suppressed osteoblast differentiation by an intracellular mechanism. A yeast two‐hybrid screen of an osteosarcoma library was conducted to identify intracellular binding partners to account for IGFBP‐6 inhibitory effects on osteoblast differentiation. LIM mineralizing protein (LMP‐1) was identified as a high affinity IGFBP‐6 binding partner. Physical interaction between IGFBP‐6 and LMP‐1 was confirmed by co‐immunoprecipitation. Fluorescent protein fusion constructs for LMP‐1 and IGFBP‐6 were transiently transfected into osteoblasts to provide evidence of subcellular locations for each protein. Coexpression of LMP‐1‐GFP and IGFBP‐6‐RFP resulted in overlapping subcellular localization of LMP‐1 and IGFBP‐6. To determine if there was a functional association of IGFBP‐6 and LMP‐1 as well as a physical association, we studied the effect of IGFBP‐6, LMP‐1 and their combination on type I procollagen promoter activity. LMP‐1 increased promoter activity while IGFBP‐6 reduced promoter activity, and coexpression of LMP‐1 with IGFBP‐6 abrogated IGFBP‐6 suppression. These studies provide evidence that overexpression of IGFBP‐6 suppresses human and murine osteoblast differentiation, that IGFBP‐6 and LMP‐1 physically interact, and supports the conclusion that this interaction may be functionally relevant. J. Cell. Biochem. 104: 1890–1905, 2008.

The diagnosis and treatment of osteoporosis: future prospects

Osteoporosis is a common disease that affects millions of patients throughout the world. We anticipate that both the diagnosis and the treatment of this disease will be revolutionized by the integration of genomics and informatics. It is predicted that a genetic algorithm will be developed to identify at-risk patients before they develop osteoporosis, so that preventive measures can be instituted. The sequencing of the human genome will lead to revolutionary advances in at least three areas of osteoporosis therapy: small molecule therapy, protein therapy and gene therapy. One area of focus for future therapeutics in osteoporosis will be on osteogenic agents, which should have a high likelihood of success because the skeleton has the innate capacity to regenerate itself.