T Lymphocytes Amplify the Anabolic Activity of Parathyroid Hormone through Wnt10b Signaling

SummaryIntermittent administration of parathyroid hormone (iPTH) is used to treat osteoporosis because it improves bone architecture and strength, but the underlying cellular and molecular mechanisms are unclear. Here, we show that iPTH increases the production of Wnt10b by bone marrow CD8+ T cells and induces these lymphocytes to activate canonical Wnt signaling in preosteoblasts. Accordingly, in responses to iPTH, T cell null mice display diminished Wnt signaling in preosteoblasts and blunted osteoblastic commitment, proliferation, differentiation, and life span, which result in decreased trabecular bone anabolism and no increase in strength. Demonstrating the specific role of lymphocytic Wnt10b, iPTH has no anabolic activity in mice lacking T-cell-produced Wnt10b. Therefore, T-cell-mediated activation of Wnt signaling in osteoblastic cells plays a key permissive role in the mechanism by which iPTH increases bone strength, suggesting that T cell osteoblast crosstalk pathways may provide pharmacological targets for bone anabolism

Exploring the associations between microRNA expression profiles and environmental pollutants in human placenta from the National Children's Study (NCS)

The placenta is the principal regulator of the in utero environment, and disruptions to this environment can result in adverse offspring health outcomes. To better characterize the impact of in utero perturbations, we assessed the influence of known environmental pollutants on the expression of microRNA (miRNA) in placental samples collected from the National Children's Study (NCS) Vanguard birth cohort. This study analyzed the expression of 654 miRNAs in 110 term placentas. Environmental pollutants measured in these placentas included dichlorodiphenyldichloroethylene (DDE), bisphenol A (BPA), polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), arsenic (As), mercury (Hg), lead (Pb), and cadmium (Cd). A moderated t-test was used to identify a panel of differentially expressed miRNAs, which were further analyzed using generalized linear models. We observed 112 miRNAs consistently expressed in >70% of the samples. Consistent with the literature, miRNAs located within the imprinted placenta-specific C19MC cluster, specifically mir-517a, mir-517c, mir-522, and mir-23a, are among the top expressed miRNA in our study. We observed a positive association between PBDE 209 and miR-188–5p and an inverse association between PBDE 99 and let-7c. Both PCBs and Cd were positively associated with miR-1537 expression level. In addition, multiple let-7 family members were downregulated with increasing levels of Hg and Pb. We did not observe DDE or BPA levels to be associated with placental miRNA expression. This is the first birth cohort study linking environmental pollutants and placental expression of miRNAs. Our results suggest that placental miRNA profiles may signal in utero exposures to environmental chemicals

The Prognostic Role and Relationship between E2F1 and SV40 in Diffuse Large B-Cell Lymphoma of Egyptian Patients

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphomas worldwide. The pathogenesis of lymphomas is not yet well understood. SV40 induces malignant transformation by the large T-antigen (L-TAG) and promotes transformation by binding and inactivating p53 and pRb. L-TAG can bind pRb promoting the activation of the E2F1 transcription factor, thus inducing the expression of genes required for the entry to the S phase and leading to cell transformation. This immunohistochemical study was conducted to assess the prognostic role and relationship of SV40 L-TAG and E2F1 in diffuse large B-cell lymphoma (DLBCL) of Egyptian patients. This retrospective study was conducted on 105 tissue specimens including 20 follicular hyperplasia and 85 DLBCL cases. SV40 L-TAG was identified in 3/85 (4%) of DLBCL. High Ki-67 labeling index (Ki-67 LI) and apoptotic count were associated with high E2F1 expression (p<0.001 for all). No significant association was reached between E2F1 and SV40. E2F1 expression proved to be the most and first independent prognostic factor on overall survival of DLBCL patients (HR = 5.79, 95% CI = 2.3–14.6, and p<0.001). Upregulation of E2F1 has been implicated in oncogenesis, prognosis, and prediction of therapeutic response but is not seemingly to have a relationship with the accused SV40

Identification of the homeobox protein, Prx1 (MHox), as a regulator of osterix expression and mediator of TNF action

Tumor necrosis factor a (TNF-a) promotes bone loss and inhibits bone formation. Osterix (Osx, SP7) is a transcription factor required for osteoblast (OB) differentiation because deletion results in a cartilaginous skeleton. We previously described a TNF suppressor element in the Osx promoter that was used to isolate nuclear proteins mediating TNF inhibition of OB differentiation. Nuclear extracts from TNF-treated pre-OBs were incubated with the TNF suppressor element for protein pull-down, and tryptic fragments were analyzed by mass spectrometry. Chromatin immunoprecipitation (ChIP) assay confirmed eight bound transcription factors. One protein, the paired related homeobox protein (Prx1), had been shown previously to have a critical role in limb bud formation and skeletal patterning. PCR revealed Prx1 expression in primary stromal cells (MSCs), C3H10T1/2 cells, and MC3T3 preosteoblasts. TNF stimulated a 14-fold increase in mRNA for Prx1, rapid cell accumulation in MC3T3 cells, and expression in periosteal and trabecular lining cells in vivo. Transient expression of Prx inhibited transcription of Osx and RUNX2. Expression of the Prx1b isoform or Prx2 decreased Osx and RUNX2 mRNA and OB differentiation in preosteoblasts. Silencing of Prx1 with siRNA abrogated TNF suppression of Osx mRNA and increased basal Osx expression. Electrophoretic mobility shift revealed Prx1b as the preferred isoform binding the Osx promoter. These results identify the homeobox protein Prx1 as an obligate mediator of TNF inhibition of Osx and differentiation of OB progenitors. Activation of Prx1 by TNF may contribute to reduced bone formation in inflammatory arthritis, menopause, and aging

Prostate carcinoma cells that have resided in bone have an upregulated IGF-I axis.

BACKGROUND: Prostate cancer (PC) has a propensity to metastasize to the skeleton, inducing an osteoblastic response in the host. Recent epidemiological studies have suggested that circulating IGF-I may be important for both the pathogenesis and dissemination of PC. We have postulated that tumor secreted IGF-I in conjunction with endogenous IGF-I contributes to the osteoblastic phenotype characteristic of metastatic PC. METHODS: To test this thesis we studied the established LNCaP PC progression model consisting of three genetically related human PC cell lines. RESULTS: Using RIA, we found serum-free conditioned media (CM) of LNCaP and C4-2 had no measurable IGF-I, whereas IGF-I was easily detected in CM from C4-2B cells at 24 hr (i.e., 1.8 +/- 0.53 ng/mg cell protein). Real-time PCR of IGF-I mRNA showed that C4-2B expressed 100-fold more IGF-I mRNA than LNCaP cells. In addition, C4-2B expression of IGF-I mRNA was substantially increased in the presence of exogenous IGF-I to nearly twofold. While IGFBP-3 and IGFBP-1 were not detectable in the CM of any PC line, all cells secreted IGFBP-2. C4-2B cells produced 40% more IGFBP-2 than LNCaP or C4-2 cells (C4-2B at 167 +/- 43 ng/mg cell protein). RANKL, a product of bone stromal cells, was also differentially expressed: LNCaP had threefold higher RANKL mRNA compared to C4-2 and C4-2B and at least equivalent protein expression. CONCLUSIONS: Our results suggest that PC cells that have metastasized to bone have an upregulated IGF-I regulatory system. This suggests an activated IGF-I axis contributes to the host-PC interaction in promoting osteoblastic metastases

Nitric oxide regulates receptor activator of nuclear factor-kappaB ligand and osteoprotegerin expression in bone marrow stromal cells.

Bone remodeling reflects an equilibrium between bone resorption and formation. The local expression of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in bone determines the entry of monoblastic precursors into the osteoclast lineage and subsequent bone resorption. Nitric oxide (NO) inhibits osteoclastic bone resorption in vitro and regulates bone remodeling in vivo. An interaction of NO with RANKL and OPG has not been studied. Here, we show that treatment of ST-2 murine stromal cells with the NO donor sodium nitroprusside (100 microm) for 24 h inhibited 1,25 dihydroxyvitamin D(3)-induced RANKL mRNA to less than 33 +/- 7% of control level, whereas OPG mRNA increased to 204 +/- 19% of control. NOR-4 replicated these NO effects. The effects of NO were dose dependent and associated with changes in protein levels: RANKL protein decreased and OPG protein increased after treatment with NO. PTH-induced RANKL expression in primary stromal cells was inhibited by sodium nitroprusside, indicating that the NO effect did not require vitamin D. NO donor did not change the stability of RANKL or OPG mRNAs, suggesting that NO affected transcription. Finally, cGMP, which can function as a second messenger for NO, did not reproduce the NO effect, nor did inhibition of endogenous guanylate cyclase prevent the NO effect on these osteoactive genes. The effect of NO to decrease the RANKL/OPG equilibrium should lead to decreased recruitment of osteoclasts and positive bone formation. Thus, drugs and conditions that cause local increase in NO formation in bone may have positive effects on bone remodeling

IGF-I regulates osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand in vitro and OPG in vivo.

IGF-I, a ubiquitous polypeptide, plays a key role in longitudinal bone growth and acquisition. The most predominant effect of skeletal IGF-I is acceleration of the differentiation program for osteoblasts. However, in vivo studies using recombinant human (rh) IGF-I and/or rhGH have demonstrated stimulation of both bone formation and resorption, thereby potentially limiting the usefulness of these peptides in the treatment of osteoporosis. In this study, we hypothesized that IGF-I modulates bone resorption by regulating expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) in bone cells. Using Northern analysis in ST2 cells, we found that human IGF-I suppressed OPG mRNA in a time- and dose-dependent manner: 100 micro g/LIGF-I (13 nM) decreased OPG expression by 37.0 +/- 1.8% (P \u3c 0.002). The half maximal inhibitory dose of IGF-I was reached at 50 micro g/liter ( approximately 6.5 nM) with no effect of IGF-I on OPG message stability. Conditioned media from ST2 cells confirmed that IGF-I decreased secreted OPG, reducing levels by 42%, from 12.1-7 ng/ml at 48 h (P \u3c 0.05). Similarly, IGF-I at 100 micro g/liter (13 nM) increased RANKL mRNA expression to 353 +/- 74% above untreated cells as assessed by real-time PCR. In vivo, low doses of rhGH when administered to elderly postmenopausal women only modestly raised serum IGF-I (to concentrations of 18-26 nM) and did not affect circulating OPG concentrations; however, administration of rhIGF-I (30 micro g/kg.d) for 1 yr to older women resulted in a significant increase in serum IGF-I (to concentrations of 39-45 nM) and a 20% reduction in serum OPG (P \u3c 0.05). In summary, we conclude that IGF-I in a dose- and time-dependent manner regulates OPG and RANKL in vitro and in vivo. These data suggest IGF-I may act as a coupling factor in bone remodeling by activating both bone formation and bone resorption; the latter effect appears to be mediated through the OPG/RANKL system in bone

IGF-I secretion by prostate carcinoma cells does not alter tumor-bone cell interactions in vitro or in vivo.

BACKGROUND: IGF-I is an important growth and differentiative factor for osteoblasts and may have a role in defining prostate cancer risk and skeletal metastases. METHODS: Conditioned media (CM) from human prostate cancer (PC), C4-2 and C4-2B, which produce osteoblastic lesions, and PC-3, which causes osteolysis, was added to MC3T3-E1 bone cultures. SCID mice were injected intratibially with these engineered cells. Tumor bearing tibiae were analyzed by microCT and pQCT. RESULTS: CM from PC cells increased osteoblast proliferation and differentiation and was unaltered by the type of PC cell, IGF-I antibodies, or exogenous IGF-I and IGFBP2. Study of intratibial PC tumors in SCID mice showed that C4-2 cells grew slowly preserving bone structure, while PC-3 tumors caused rapid osteolysis. Overexpression of IGF-I did not change either tumor progression or skeletal response. CONCLUSIONS: IGF-I is neither necessary nor sufficient for the osteoblastic response to PC metastases. Prostate (c) 2006 Wiley-Liss, Inc