bluuebunny/crossref_metadata_2025_split huggingface dataset:
Growth Hormone Attenuates the Transcriptional Activity of Runx2 by Facilitating Its Physical Association With Stat3β
Authors: Ziros Panos G , Georgakopoulos Tassos , Habeos Ioannis , Basdra Efthimia K , Papavassiliou Athanasios G 
Published: April 2006

Abstract
We document that GH controls osteoblast function by modulating the biological activity of the osteospecific transcription factor Runx2. Evidence is provided for a physical interaction between Runx2 and Stat3β, which is enhanced by GH and downregulates the transcriptional properties of this key osteogenic regulator.
Introduction: Growth hormone (GH) signals to bone either through insulin-like growth factor-1 or directly by influencing the function of osteoblasts, the bone-forming cells. This study aimed at exploring the molecular events that underlie the direct biological action of GH on osteoblastic cells, and specifically, the effects that it might exert on the function of the bone-specific transcriptional regulator Runx2.
Materials and Methods: The GH-responsive human osteoblastic cell line Saos-2 was used as our experimental system. Western blot analyses were used to monitor the presence of several parameters known to be affected by GH in these cells (i.e., downregulation of GH receptor, induction of STATs, and extracellular signal-regulated kinase [ERK] mitogen-activated protein kinase [MAPK] pathways). Electrophoretic mobility shift assays were used to assess Runx2 and Stat3 binding activity on an osteoblast-specific element (OSE2) after GH treatment. A combination of yeast two-hybrid and co-immunoprecipitation assays were performed to test for the existence of a physical Runx2·Stat3β association. Finally, co-transfection experiments were used to investigate the interplay of the two transcription factors on the activity of a p6OSE2-Luc promoter after GH stimulation.
Results: We show that GH signaling through Stat3/ERK MAPK potentiates the DNA binding activity of Runx2 but, at the same time, restrains its transcriptional potential. Moreover, a novel physical interaction of Runx2 with transcription factor Stat3β, which is enhanced by GH stimulation, was documented both in vitro and in vivo. Importantly, this interaction impairs the transcriptional activity of Runx2 without affecting its DNA binding capacity.
Conclusion: Our data provide the first evidence that GH modulates the transcriptional function of Runx2 in osteoblastic cells by promoting its inhibitory interaction with Stat3β. Shedding light on such mechanisms will contribute to a better understanding of GH effects on skeletal homeostasis that may impact on decisions at the clinical level, especially in diseases affecting bone quantity and quality (e.g., osteoporosis).
Interleukin-1β and TNF-α Act in Synergy to Inhibit Longitudinal Growth in Fetal Rat Metatarsal Bones
Authors: Mårtensson Katarina , Chrysis Dionisios , Sävendahl Lars 
Published: April 2006

Abstract
We hypothesized that pro-inflammatory cytokines can act locally in the growth plate to impair longitudinal growth. In a model of cultured fetal rat metatarsal bones, we found that IL-1β and TNF-α act in synergy to inhibit longitudinal growth, an effect linked to decreased proliferation and increased apoptosis of growth plate chondrocytes. IGF-I could partially reverse all these effects.
Introduction: Children with chronic inflammatory conditions, such as Crohn's disease or rheumatoid arthritis, experience impaired longitudinal growth. The inflammatory process itself, which includes upregulation of the pro-inflammatory cytokines interleukin (IL)-1β, IL-6, and TNF-α, is believed to be at least partly responsible for the poor growth in these patients. This study aimed to clarify whether these cytokines can act locally in the growth plate to suppress longitudinal growth and whether any negative effects can be reversed by insulin-like growth factor-I (IGF-I).
Materials and Methods: The effects of cytokines on longitudinal bone growth were studied in fetal (day E20) rat metatarsal bones kept in culture. After a 7-day culture, the bones were sectioned, and chondrocyte proliferation was assessed by bromodeoxyuridine (BrdU) incorporation and apoptosis by TUNEL.
Results: When added separately, IL-1β and TNF-α impaired longitudinal bone growth only at a high concentration (100 ng/ml each; p &lt; 0.05 versus control). In contrast, when added in combination, IL-1β and TNF-α potently inhibited growth at far lower concentrations (from 3 ng/ml each; p &lt; 0.001 versus control) and also decreased chondrocyte proliferation and increased apoptosis. Growth failure induced by the combination of IL-1β and TNF-α (10 ng/ml each) could be counteracted by anti-IL-1β (100 ng/ml; p &lt; 0.001), anti-TNF-α (100 ng/ml; p &lt; 0.001), or IGF-I (100 ng/ml; p &lt; 0.01). IL-6 did not affect longitudinal growth even when added in combination with IL-1β or TNF-α (10 ng/ml each).
Conclusions: We show that IL-1β and TNF-α act in synergy to locally suppress longitudinal growth, an effect that can be partially reversed by IGF-I. Although growth hormone (GH)/IGF-I may improve longitudinal growth in children with chronic inflammatory diseases, our results suggest that the inflammatory process itself must be targeted to achieve normal growth.
Downregulation of Osteoblast Markers and Induction of the Glial Fibrillary Acidic Protein by Oncostatin M in Osteosarcoma Cells Require PKCδ and STAT3
Authors: Chipoy Céline , Berreur Martine , Couillaud Séverine , Pradal Gilbert , Vallette François , Colombeix Caroline , Rédini Françoise , Heymann Dominique , Blanchard Frédéric 
Published: April 2006

Abstract
The effects of OSM on proliferation and differentiation of osteosarcoma and nontransformed osteoblasts were analyzed. OSM downregulates osteoblast markers but induces the glial fibrillary acidic protein by the combined activation of PKCδ and STAT3, offering new lines of therapeutic investigations.
Introduction: Oncostatin M (OSM) is a multifunctional cytokine of the interleukin-6 family implicated in embryonic development, differentiation, inflammation, and regeneration of various tissues, mainly the liver, bone, and the central nervous and hematopoietic systems. One particularity of OSM relies on its growth inhibitory and pro-differentiating effects on a variety of tumor cell lines such as melanoma, providing arguments for a therapeutic application of OSM. The objective of this study was to analyze the effects of OSM on osteosarcoma cell lines proliferation and differentiation.
Materials and Methods: Proliferation was analyzed by3H thymidine incorporation. Differentiation was analyzed by semiquantitative RT-PCR and immunocytochemistry for various markers. Alizarin red S staining was used to evaluate bone nodule formation. Morphological changes were studied by confocal and electron microscopy. Western blotting, kinases inhibitors, and dominant negative STAT3 were used to identified the signaling pathways implicated.
Results: OSM inhibits the growth of rat osteosarcoma cell lines as well as normal osteoblasts, in correlation with induction of the cyclin-dependent kinases inhibitor p21WAF1. However, OSM reduces osteoblast markers such as alkaline phosphatase, osteocalcin, and bone sialoprotein, leading to strong inhibition of mineralized nodule formation. This inhibitory effect is restricted to mature osteoblasts and differentiated osteosarcoma because OSM effectively stimulates osteoblast markers and bone nodule formation in early, but not late, bone marrow mesenchymal stem cell (BMSC) cultures. In osteosarcoma cells or BMSC, OSM induces expression of the glial fibrillary acidic protein (GFAP) as well as morphological and ultrastructural changes, for example, elongated shape and bundles of microfilaments in cell processes. Rottlerin (PKCδ inhibitor), and to a lesser degree UO126 (MEK/ERK inhibitor), prevents the loss of osteoblastic markers by OSM, whereas dominant negative STAT3 prevents GFAP induction.
Conclusions: These results highlight the particular gene expression profile of OSM-treated osteosarcoma cells and BMSCs, suggesting either a osteocytic or a glial-like phenotype. Together with the implication of PKCδ, ERK1/2, and STAT3, these results offer new lines of investigations for neural cell transplantation and osteosarcoma therapy.
Quantitative Trait Loci That Determine BMD in C57BL/6J and 129S1/SvImJ Inbred Mice
Authors: Ishimori Naoki , Li Renhua , Walsh Kenneth A , Korstanje Ron , Rollins Jarod A , Petkov Petko , Pletcher Mathew T , Wiltshire Tim , Donahue Leah Rae , Rosen Clifford J , Beamer Wesley G , Churchill Gary A , Paigen Beverly 
Published: April 2006

Abstract
BMD is highly heritable; however, little is known about the genes. To identify loci controlling BMD, we conducted a QTL analysis in a (B6 × 129) F2 population of mice. We report on additional QTLs and also narrow one QTL by combining the data from multiple crosses and through haplotype analysis.
Introduction: Previous studies have identified quantitative trait loci (QTL) that determine BMD in mice; however, identification of genes underlying QTLs is impeded by the large size of QTL regions.
Materials and Methods: To identify loci controlling BMD, we performed a QTL analysis of 291 (B6 × 129) F2 females. Total body and vertebral areal BMD (aBMD) were determined by peripheral DXA when mice were 20 weeks old and had consumed a high-fat diet for 14 weeks.
Results and Conclusions: Two QTLs were common for both total body and vertebral aBMD: Bmd20 on chromosome (Chr) 6 (total aBMD; peak cM 26, logarithm of odds {LOD} 3.8, and vertebral aBMD; cM 32, LOD 3.6) and Bmd22 on Chr 1 (total aBMD; cM 104, LOD 2.5, and vertebral aBMD; cM 98, LOD 2.6). A QTL on Chr 10 (Bmd21, cM 68, LOD 3.0) affected total body aBMD and a QTL on Chr 7 (Bmd9, cM 44, LOD 2.7) affected vertebral aBMD. A pairwise genome-wide search did not reveal significant gene-gene interactions. Collectively, the QTLs accounted for 21.6% of total aBMD and 17.3% of vertebral aBMD of the F2 population variances. Bmd9 was previously identified in a cross between C57BL/6J and C3H/HeJ mice, and we narrowed this QTL from 34 to 22 cM by combining the data from these crosses. By examining the Bmd9 region for conservation of ancestral alleles among the low allele strains (129S1/SvImJ and C3H/HeJ) that differed from the high allele strain (C57BL/6J), we further narrowed the region to ∼9.9 cM, where the low allele strains share a common haplotype. Identifying the genes for these QTLs will enhance our understanding of skeletal biology.
Four and Half Lim Protein 2 (FHL2) Stimulates Osteoblast Differentiation
Authors: Lai Chung-Fang , Bai Shuting , Uthgenannt Brian A , Halstead Linda R , McLoughlin Patricia , Schafer Beat W , Chu Po-Hsien , Chen Ju , Otey Carol A , Cao Xu , Cheng Su-Li 
Published: April 2006

Abstract
FHL2, a molecule that interacts with many integrins and transcription factors, was found to play an important role in osteoblast differentiation. Overexpression of FHL2 increases the accumulation of osteoblast differentiation markers and matrix mineralization, whereas FHL2 deficiency results in inhibition of osteoblast differentiation and decreased bone formation.
Introduction: Integrin-matrix interaction plays a critical role in osteoblast function. It has been shown that the cytoplasmic domains of integrin β subunits mediate signal transduction induced by integrin-matrix interaction. We reasoned that the identification of proteins interacting with β-cytoplasmic tails followed by analysis of the function of these proteins would enhance our understanding on integrin signaling and the roles of these proteins in osteoblast activities.
Materials and Methods: Yeast two hybrid assay was used to identify proteins interacting with the cytoplasmic domain of integrin β5 subunit. The association of these proteins with integrin αvβ5 was confirmed by confocal analysis and co-immunoprecipitation. A stable MC3T3-E1 cells line overexpressing Four and Half Lim Protein 2 (FHL2) and mouse osteoblasts deficient in FHL2 were used to study the roles of FHL2 in osteoblast differentiation and bone formation. Matrix protein expression was determined by mRNA analysis and Western blotting. Matrix mineralization was detected by Alizarin red staining. Alkaline phosphatase activity was also measured. μCT was used to determine bone histomorphometry.
Results and Conclusions: FHL2 and actin-binding proteins, palladin and filamin A, were identified as proteins interacting with β5 cytoplasmic domain. FHL2 co-localized with αvβ5 at the focal adhesion sites in association with palladin and filamin A. FHL2 was also present in nuclei. Osteoblasts overexpressing FHL2 exhibited increased adhesion to and migration on matrix proteins. Conversely, FHL2 stimulation of CREB activity was dependent on integrin function because it was inhibited by Gly-Arg-Gly-Asp-Ser (GRGDS) peptide. The expression of osteoblast differentiation markers and Msx2 was upregulated, and bone matrix mineralization was increased in FHL2 overexpressing cells. In contrast, FHL2-deficient bone marrow cells and osteoblasts displayed decreased osteoblast colony formation and differentiation, respectively, compared with wildtype cells. Moreover, FHL2-deficient female mice exhibited greater bone loss than the wildtype littermates after ovariectomy. Thus, FHL2 plays an important role in osteoblast differentiation and bone formation.
TREM2, a DAP12-Associated Receptor, Regulates Osteoclast Differentiation and Function
Authors: Humphrey Mary Beth , Daws Michael R , Spusta Steve C , Niemi Eréne C , Torchia James A , Lanier Lewis L , Seaman William E , Nakamura Mary C 
Published: April 2006

Abstract
Deficiency of the signaling adapter protein DAP12 or its associated receptor TREM2 is associated with abnormal OC development in humans. Here we examine the role of TREM2 in mouse OC development and function, including migration and resorption in vitro. These results provide new evidence that TREM2 regulates OC function independent of its effects on multinucleated OC differentiation.
Introduction: TREM2 (triggering receptor expressed in myeloid cells-2) associates with the signaling adapter DAP12 in osteoclasts (OCs). Genetic mutation or deletion of either the TYROBP (DAP12) or TREM2 gene is associated with the human disorder of brain and bone, Nasu-Hakola disease. We and others recently showed the critical requirement for immunoreceptor tyrosine-based activation motif (ITAM) signals through DAP12 and the Fc Receptor γ chain (FcRγ) during OC development. Here, we further define the role of TREM2 in OC differentiation and describe a role for TREM2 in OC migration and bone resorption.
Materials and Methods: We generated monoclonal anti-mouse TREM2 antibodies (mAb), analyzed pre-osteoclasts and mature OCs for TREM2 surface expression, and determined the effect of antibody ligation on in vitro OC differentiation, resorption, and migration. TREM2 RNA interference (RNAi) was used to disrupt expression of TREM2 in pre-osteoclasts.
Results: Using flow cytometry, our studies reveal that TREM2 is weakly expressed on C57BL/6 bone marrow macrophages (BMMs) and is upregulated during culture with RANKL and macrophage-colony stimulating factor (M-CSF). The expression of TREM2 is unaltered in DAP12-deficient OCs. Using C57BL/6 BMMs or RAW264.7 precursors, anti-TREM2 mAb treatment with RANKL and M-CSF enhances the formation of multinuclear TRACP+ OCs compared with control mAb treatment. In contrast, these agents have no effect on DAP12-deficient precursors. Monoclonal Ab blockade of TREM2 on OCs generated from C57BL/6 BMMs results in decreased resorption of artificial calcium-phosphate substrate and dentine. Reduction of TREM2 expression in RAW264.7 cells by RNAi results in loss of OC formation in response to RANKL and M-CSF. Anti-TREM2 cross-linking enhances migration of C57BL/6 OCs and RAW246.7 OCs in response to M-CSF.
Conclusions: Our studies indicate that the TREM2 receptor regulates OC multinucleation as well as resorption and migration of mature OCs. Thus, TREM2-DAP12 signals regulate both OC formation and function.
Gorham-Stout Syndrome: A Monocyte-Mediated Cytokine Propelled Disease
Authors: Colucci Silvia , Taraboletti Giulia , Primo Luca , Viale Andrea , Roca Cristina , Valdembri Donatella , Geuna Massimo , Pagano Marco , Grano Maria , Pogrel Anthony M , Harris Adrian L , Athanasou Nicholas N , Mantovani Alberto , Zallone Alberta , Bussolino Federico 
Published: April 2006

AbstractWe studied the biological features and the immunophenotype of a cell culture established from the lesion of soft tissues of a woman affected by Gorham-Stout syndrome. We found that these cells belonged to a monocytic lineage with some characteristics of immature osteoclasts and were able to release large amounts of osteoclastogenic and angiogenic molecules that may contribute to disease progression.Introduction: Gorham-Stout syndrome is a rare disease characterized by osteolysis and proliferation of vascular or lymphatic vessels, with a severe outcome. Its etiology and the identification of the cell types involved are completely unknown.Materials and Methods: A cell culture from a lesion of soft tissues was established, and its behavior in vitro and in immunodeficient mice was studied. We analyzed (1) the cell phenotype by flow cytometry; (2) the adhesive and migratory properties on different substrates; (3) the ability to differentiate into mature osteoclasts; (4) the production of osteclastogenic and angiogenic molecules; (5) the in vivo angiogenic activity of the cells subcutaneously implanted in mouse in a Matrigel plug; and (6) the ability to recapitulate the disease when transplanted in nude mice.Results and Conclusions: The established culture consisted of a morphologically homogeneous cell population belonging to a monocytic lineage having some features of an osteoclast-like cell type. Cells had an invasive phenotype, were angiogenic, and produced osteoclastogenic (IL-6, TGF-β1, IL-1β) and angiogenic (vascular endothelial growth factor-A {VEGF-A}, CXCL-8) molecules when challenged with inflammatory cytokines. Immunodeficient mice injected with these cells did not show any bone lesions or vascular alteration, but had high amounts of circulating human IL-6 and VEGF-A. Cells isolated from a cutaneous lymphangiomatosis did not show any of these findings. These data suggest that cells of monocyte-macrophage lineage play an essential role in the pathogenesis of Gorham-Stout disease, whose progression is propelled by cytokine circuits that accelerate angiogenesis and osteoclastogenesis.
Effect of Teriparatide {rhPTH(1-34)} on BMD When Given to Postmenopausal Women Receiving Hormone Replacement Therapy
Authors: Ste-Marie Louis G , Schwartz Sherwyn L , Hossain Anwar , Desaiah Durisala , Gaich Gregory A 
Published: April 2006

Abstract
The effects of teriparatide when given in combination with HRT were studied in postmenopausal women with low bone mass or osteoporosis. The data provide evidence that the adverse event profile for combination therapy with teriparatide + HRT together is consistent with that expected for each treatment alone and that the BMD response is greater than for HRT alone.
Introduction: Teriparatide {rhPTH(1-34)}, given as a once-daily injection, activates new bone formation in patients with osteoporosis. Hormone replacement therapy (HRT) prevents osteoporosis by reducing bone resorption and formation. Combination therapy with these two compounds, in small clinical trials, increased BMD and reduced vertebral fracture burden. The purpose of this study was to determine whether teriparatide provided additional effect on BMD when given in combination with HRT.
Materials and Methods: A randomized, double-blind, placebo-controlled study was conducted in postmenopausal women with either low bone mass or osteoporosis. Patients were randomized to placebo subcutaneous plus HRT (n = 125) or teriparatide 40 μg/day (SC) plus HRT (TPTD40 + HRT; n = 122) for a median treatment exposure of 13.8 months. Approximately one-half of the patients in each group were pretreated with HRT for at least 12 months before randomization. Patients received 1000 mg calcium and 400–1200 IU of vitamin D daily as oral supplementation. BMD was measured by DXA.
Results: Compared with HRT alone, TPTD40 + HRT produced significant (p &lt; 0.001) increases in spine BMD (14% versus 3%), total hip (5.2% versus 1.6%), and femoral neck (5.2% versus 2%) at study endpoint. BMD, in whole body and ultradistal radius, was higher, and in the one-third distal radius was lower, in the combination therapy but not in the HRT group. Serum bone-specific alkaline phosphatase and urinary N-telopeptide/Cr were increased significantly (p &lt; 0.01) in the women receiving TPTD40 + HRT compared with HRT. A similar profile of BMD and bone markers was evident in both randomized patients as well as in subgroups of patients not pretreated or pretreated with HRT. Patients tolerated both the treatments well. Nausea and leg cramps were more frequently reported in the TPTD40 + HRT group.
Conclusions: Adding teriparatide, a bone formation agent, to HRT, an antiresorptive agent, provides additional increases in BMD beyond that provided by HRT alone. The adverse effects of teriparatide when added to HRT were similar to the adverse effects described for teriparatide administered alone. Whether teriparatide was initiated at the same time as HRT or after at least 1 year on HRT, the incremental increases over HRT alone were similar.