Stein, Stein, Lian, vanWijnen Lab Copyright (c) 2013 University of Massachusetts Medical School All rights reserved. http://escholarship.umassmed.edu/stein Recent documents in Stein, Stein, Lian, vanWijnen Lab en-us Wed, 13 Feb 2013 18:51:21 PST 3600 Forum on aging and skeletal health: summary of the proceedings of an ASBMR workshop http://escholarship.umassmed.edu/stein/265 http://escholarship.umassmed.edu/stein/265 Fri, 11 May 2012 07:55:56 PDT With the aging of the population, the scope of the problem of age-related bone loss and osteoporosis will continue to increase. As such, it is critical to obtain a better understanding of the factors determining the acquisition and loss of bone mass from childhood to senescence. While there have been significant advances in recent years in our understanding of both the basic biology of aging and a clinical definition of age-related frailty, few of these concepts in aging research have been evaluated adequately for their relevance and application to skeletal aging or fracture prevention. The March 2011 Forum on Aging and Skeletal Health, sponsored by the NIH and ASBMR, sought to bring together leaders in aging and bone research to enhance communications among diverse fields of study so as to accelerate the pace of scientific advances needed to reduce the burden of osteoporotic fractures. This report summarizes the major concepts presented at that meeting and in each area identifies key questions to help set the agenda for future research in skeletal aging.

]]> Sundeep Khosla et al. Aging *Biomedical Research Bone Resorption Bone and Bones Cell Aging Fractures, Bone Genetic Predisposition to Disease *Health Humans Minerals Motor Activity *Societies, Scientific Neural Crest Deletion of Dlx3 Leads to Major Dentin Defects through Down-regulation of Dspp http://escholarship.umassmed.edu/stein/264 http://escholarship.umassmed.edu/stein/264 Fri, 11 May 2012 07:55:51 PDT During development, Dlx3 is expressed in ectodermal appendages such as hair and teeth. Thus far, the evidence that Dlx3 plays a crucial role in tooth development comes from reports showing that autosomal dominant mutations in DLX3 result in severe enamel and dentin defects leading to abscesses and infections. However, the normal function of DLX3 in odontogenesis remains unknown. Here, we use a mouse model to demonstrate that the absence of Dlx3 in the neural crest results in major impairment of odontoblast differentiation and dentin production. Mutant mice develop brittle teeth with hypoplastic dentin and molars with an enlarged pulp chamber and underdeveloped roots. Using this mouse model, we found that dentin sialophosphoprotein (Dspp), a major component of the dentin matrix, is strongly down-regulated in odontoblasts lacking Dlx3. Using ChIP-seq, we further demonstrate the direct binding of Dlx3 to the Dspp promoter in vivo. Luciferase reporter assays determined that Dlx3 positively regulates Dspp expression. This establishes a regulatory pathway where the transcription factor Dlx3 is essential in dentin formation by directly regulating a crucial matrix protein.

]]> Olivier Duverger et al. Transcription Factors Homeodomain Proteins Dentin Neural Crest Odontoblasts Vitamin k-dependent carboxylation of osteocalcin: friend or foe http://escholarship.umassmed.edu/stein/263 http://escholarship.umassmed.edu/stein/263 Fri, 11 May 2012 07:55:45 PDT Osteocalcin originates from osteoblastic synthesis and is deposited into bone or released into circulation, where it correlates with histological measures of bone formation. The presence of 3 vitamin K-dependent gamma carboxyglutamic acid residues is critical for osteocalcin's structure, which appears to regulate the maturation of bone mineral. In humans, the percentage of the circulating osteocalcin that is not gamma-carboxylated (percent ucOC) is used as a biomarker of vitamin K status. In contrast, when ucOC is not corrected for total osteocalcin, the interpretation of this measure is confounded by osteoblastic activity, independent of vitamin K. Observational studies using percent ucOC have led to the conclusion that vitamin K insufficiency leads to age-related bone loss. However, clinical trials do not provide overall support for the suggestion that vitamin K supplementation of the general population will reduce bone loss or fracture risk. More recently, results from in vitro and in vivo studies using animal models indicate that ucOC is an active hormone with a positive role in glucose metabolism. By inference, vitamin K, which decreases ucOC, would have a detrimental effect. However, in humans this hypothesis is not supported by the limited data available, nor is it supported by what has been established regarding osteocalcin chemistry. In summary, the specific function of osteocalcin in bone and glucose metabolism has yet to be elucidated.

]]> Caren M. Gundberg et al. Osteocalcin Vitamin K Osteoarthritic tissues modulate functional properties of sensory neurons associated with symptomatic OA pain http://escholarship.umassmed.edu/stein/262 http://escholarship.umassmed.edu/stein/262 Fri, 11 May 2012 07:55:39 PDT Osteoarthritis (OA) is an age-related degenerative disease of cartilaginous tissues that is accompanied by hyperalgesia. Molecular cause and effect relationships between OA and pain remain to be elucidated. In this study, we have developed an experimental ex vivo organ co-culture system with dorsal root ganglia (DRGs) and knee synovial tissues from OA patients or unaffected human subjects. Our results suggest that tissues may generate symptomatic pain by altering the functional properties of sensory neurons. Specifically, we find that the expression levels of genes associated with neuronal pathways (e.g., SP, NK1, NK2, NPYR1, NPYR2, alpha2delta1) or inflammation (COX2/PTGS2 and IL6/interferon beta2) are clearly elevated in DRG explants cultured in the presence of OA derived synovial tissues. These findings are consistent with a model in which cytokines and pain molecules produced by knee synovium sensitize nociceptive neurons in tissues peripheral to joint cartilage.

]]> Xin Li et al. Animals Coculture Techniques Ganglia, Spinal Gene Expression Regulation Humans Osteoarthritis Pain Rats Rats, Sprague-Dawley Sensory Receptor Cells Synovial Membrane MicroRNA-146a is linked to pain-related pathophysiology of osteoarthritis http://escholarship.umassmed.edu/stein/261 http://escholarship.umassmed.edu/stein/261 Fri, 11 May 2012 07:55:32 PDT Because miR-146a is linked to osteoarthritis (OA) and cartilage degeneration is associated with pain, we have characterized the functional role of miR-146a in the regulation of human articular cartilage homeostasis and pain-related factors. Expression of miRNA 146a was analyzed in human articular cartilage and synovium, as well as in dorsal root ganglia (DRG) and spinal cord from a rat model for OA-related pain assessment. The functional effects of miR-146a on human chondrocytic, synovial, and microglia cells were studied in cells transfected with miR-146a. Using real-time PCR, we assessed the expression of chondrocyte metabolism-related genes in chondrocytes, genes for inflammatory factors in synovial cells, as well as pain-related proteins and ion channels in microglial cells. Previous studies showed that miR-146a is significantly upregulated in human peripheral knee OA joint tissues. Transfection of synthetic miR-146a significantly suppresses extracellular matrix-associated proteins (e.g., Aggrecan, MMP-13, ADAMTS-5, collagen II) in human knee joint chondrocytes and regulates inflammatory cytokines in synovial cells from human knee joints. In contrast, miR-146a is expressed at reduced levels in DRGs and dorsal horn of the spinal cords isolated from rats experiencing OA-induced pain. Exogenous supplementation of synthetic miR-146a significantly modulates inflammatory cytokines and pain-related molecules (e.g., TNFalpha, COX-2, iNOS, IL-6, IL8, RANTS and ion channel, TRPV1) in human glial cells. Our findings suggest that miR-146a controls knee joint homeostasis and OA-associated algesia by balancing inflammatory responses in cartilage and synovium with pain-related factors in glial cells. Hence, miR-146a may be useful for the treatment of both cartilage regeneration and pain symptoms caused by OA.

]]> Xin Li et al. Animals Cartilage, Articular Cells, Cultured Chondrocytes Humans MicroRNAs Neural Pathways Neuroglia Osteoarthritis Osteoarthritis, Knee Pain Rats Synovial Membrane Transfection Lactoferricin mediates anabolic and anti-catabolic effects in the intervertebral disc http://escholarship.umassmed.edu/stein/260 http://escholarship.umassmed.edu/stein/260 Fri, 11 May 2012 07:55:26 PDT Lactoferricin (LfcinB) antagonizes biological effects mediated by angiogenic and catabolic growth factors, in addition to pro-inflammatory cytokines and chemokines in human endothelial cells and tumor cells. However, the effect of LfcinB on intervertebral disc (IVD) cell metabolism has not yet been investigated. Using bovine nucleus pulposus (NP) cells, we analyzed the effect of LfcinB on proteoglycan (PG) accumulation, PG synthesis, and anabolic gene expression. We assessed expression of genes for matrix-degrading enzymes such as matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family), as well as their endogenous inhibitors, tissue inhibitor of metalloproteases (TIMPs). In order to understand the specific molecular mechanisms by which LfcinB exerts its biological effects, we investigated intracellular signaling pathways in NP cells. LfcinB increased PG accumulation mainly via PG synthesis in a dose-dependent manner. Simultaneously, LfcinB dose-dependently downregulated catabolic enzymes. LfcinB's anti-catabolic effects were further demonstrated by a dose-dependent increase in multiple TIMP family members. Our results demonstrate that ERK and/or p38 mitogen-activated protein kinase pathways are the key signaling cascades that exert the biological effects of LfcinB in NP cells, regulating transcription of aggrecan, SOX-9, TIMP-1, TIMP-2, TIMP-3, and iNOS. Our results suggest that LfcinB has anabolic and potent anti-catabolic biological effects on bovine IVD cells that may have considerable promise in the treatment of disc degeneration in the future.

]]> Jae-Sung Kim et al. ADAM Proteins Anabolic Agents Animals Cattle Cells, Cultured Extracellular Matrix Proteins Gene Expression Humans Intervertebral Disc Intervertebral Disc Degeneration Lactoferrin MAP Kinase Signaling System Oxidative Stress Proteoglycans Signal Transduction Tissue Inhibitor of Metalloproteinases The polypyrimidine/polypurine motif in the mouse mu opioid receptor gene promoter is a supercoiling-regulatory element http://escholarship.umassmed.edu/stein/259 http://escholarship.umassmed.edu/stein/259 Fri, 11 May 2012 07:55:18 PDT The mu opioid receptor (MOR) is the principle molecular target of opioid analgesics. The polypyrimidine/polypurine (PPy/u) motif enhances the activity of the MOR gene promoter by adopting a non-B DNA conformation. Here, we report that the PPy/u motif regulates the processivity of torsional stress, which is important for endogenous MOR gene expression. Analysis by topoisomerase assays, S1 nuclease digests, and atomic force microscopy showed that, unlike homologous PPy/u motifs, the position- and orientation-induced structural strains to the mouse PPy/u element affect its ability to perturb the relaxation activity of topoisomerase, resulting in polypurine strand-nicked and catenated DNA conformations. Raman spectrum microscopy confirmed that mouse PPy/u containing-plasmid DNA molecules under the different structural strains have a different configuration of ring bases as well as altered Hoogsteen hydrogen bonds. The mouse MOR PPy/u motif drives reporter gene expression fortyfold more effectively in the sense orientation than in the antisense orientation. Furthermore, mouse neuronal cells activate MOR gene expression in response to the perturbations of topology by topoisomerase inhibitors, whereas human cells do not. These results suggest that, interestingly among homologous PPy/u motifs, the mouse MOR PPy/u motif dynamically responds to torsional stress and consequently regulates MOR gene expression in vivo.

]]> Chung-youl Choe et al. Animals Binding Sites Camptothecin Cell Line, Tumor DNA Gene Expression Regulation Humans Luciferases Mice Microscopy, Atomic Force Nucleic Acid Conformation Promoter Regions, Genetic Purine Nucleotides Pyrimidine Nucleotides Receptors, Opioid, mu Regulatory Sequences, Nucleic Acid Reverse Transcriptase Polymerase Chain Reaction Spectrum Analysis, Raman Topoisomerase I Inhibitors Characterization of a new animal model for evaluation and treatment of back pain due to lumbar facet joint osteoarthritis http://escholarship.umassmed.edu/stein/258 http://escholarship.umassmed.edu/stein/258 Fri, 11 May 2012 07:55:11 PDT OBJECTIVE: Osteoarthritic (OA) degeneration of the lumbar facet joints has been implicated in low back pain. This study was undertaken to investigate the biologic links between cellular and structural alterations within facet joint components and the development of symptomatic chronic back pain.

METHODS: We generated an animal model of facet joint degeneration by intraarticular injection of monosodium iodoacetate (MIA) into facet joints (L3-L4, L4-L5, L5-L6) of Sprague-Dawley rats. Pain sensation due to pressure, which mimics a mechanical stimulus for facet joint injury, was measured using an algometer. Pain response was also assessed in a straight leg raising test. Cartilage alterations were assessed by biochemical evaluation and microfocal computed tomography (micro-CT). Therapeutic modulation of chronic facet joint pain with the use of various pharmacologic agents was investigated.

RESULTS: MIA injection resulted in severely damaged facet joint cartilage, proteoglycan loss, and alterations of subchondral bone structure. Micro-CT analyses suggested that the behavioral hyperalgesia from facet joint degeneration was not associated with foraminal stenosis. The biologic and structural changes in facet joints were closely associated with sustained and robust chronic pain. Morphine and pregabalin markedly alleviated pressure hyperalgesia, while celecoxib (a selective inhibitor of cyclooxygenase 2 [COX-2]) produced moderate antihyperalgesic effects and the effect of ketorolac (an inhibitor of COX-1 and COX-2) was negligible.

CONCLUSION: Our findings demonstrate that MIA injection provides a useful model for the study of OA changes in the facet joint and indicate that facet joint degeneration is a major cause of chronic low back pain. The treatment results suggest that classes of drugs that are widely used to treat OA, such as nonsteroidal antiinflammatory drugs, may have limited efficacy once joint destruction is complete.

]]> Jae-Sung Kim et al. Zygapophyseal Joint Osteoarthritis, Spine Iodoacetates Control of mesenchymal lineage progression by microRNAs targeting the skeletal gene regulators Trps1 and Runx2 http://escholarship.umassmed.edu/stein/257 http://escholarship.umassmed.edu/stein/257 Mon, 07 May 2012 13:11:53 PDT Multiple microRNAs (miRNAs) that target the osteogenic Runt-related transcription factor 2 (RUNX2) define an interrelated network of miRNAs that control osteoblastogenesis. We addressed whether these miRNAs have functional targets beyond RUNX2 that co-regulate skeletal development. Here, we find that seven RUNX2-targeting miRNAs (miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-205 and miR-217) also regulate the chondrogenic GATA transcription factor Tricho-Rhino-Phalangeal Syndrome I (TRPS1). While the efficacy of each miRNA to target RUNX2 or TRPS1 differs in osteoblasts and chondrocytes, each effectively blocks maturation of pre-committed osteoblasts and chondrocytes. Furthermore, these miRNAs can redirect mesenchymal stem cells into adipogenic cell fate with concomitant up-regulation of key lineage specific transcription factors. Thus, a program of multiple miRNAs controls mesenchymal lineage progression by selectively blocking differentiation of osteoblasts and chondrocytes to control skeletal development.

]]> Ying Zhang et al. MicroRNAs Osteoblasts Chondrocytes DNA-Binding Proteins Transcription Factors Core Binding Factor Alpha 1 Subunit Mesenchymal Stem Cells Musculoskeletal Development Targeted inhibition of mitochondrial Hsp90 suppresses localised and metastatic prostate cancer growth in a genetic mouse model of disease http://escholarship.umassmed.edu/stein/256 http://escholarship.umassmed.edu/stein/256 Thu, 26 Apr 2012 08:49:10 PDT BACKGROUND: The molecular chaperone heat shock protein-90 (Hsp90) is a promising cancer drug target, but current Hsp90-based therapy has so far shown limited activity in the clinic.

METHODS: We tested the efficacy of a novel mitochondrial-targeted, small-molecule Hsp90 inhibitor, Gamitrinib (GA mitochondrial matrix inhibitor), in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. The TRAMP mice receiving 3-week or 5-week systemic treatment with Gamitrinib were evaluated for localised or metastatic prostate cancer, prostatic intraepithelial neoplasia (PIN) or localised inflammation using magnetic resonance imaging, histology and immunohistochemistry. Treatment safety was assessed histologically in organs collected at the end of treatment. The effect of Gamitrinib on mitochondrial dysfunction was studied in RM1 cells isolated from TRAMP tumours.

RESULTS: Systemic administration of Gamitrinib to TRAMP mice inhibited the formation of localised prostate tumours of neuroendocrine or adenocarcinoma origin, as well as metastatic prostate cancer to abdominal lymph nodes and liver. The Gamitrinib treatment had no effect on PIN or prostatic inflammation, and caused no significant animal weight loss or organ toxicity. Mechanistically, Gamitrinib triggered acute mitochondrial dysfunction in RM1 cells, with loss of organelle inner membrane potential and release of cytochrome-c in the cytosol.

CONCLUSIONS: The Gamitrinib has pre-clinical activity and favourable tolerability in a genetic model of localised and metastatic prostate cancer in immunocompetent mice. Selective targeting of mitochondrial Hsp90 could provide novel molecular therapy for patients with advanced prostate cancer.

]]> Byoung H. Kang et al. Adenocarcinoma Animals Antineoplastic Agents Cell Proliferation Cells, Cultured Disease Models, Animal Disease Progression Drug Evaluation, Preclinical Female Genetic Predisposition to Disease Guanidines HSP90 Heat-Shock Proteins Lactams, Macrocyclic Male Mice Mice, Inbred C57BL Mice, Transgenic Mitochondria Molecular Targeted Therapy Neoplasm Metastasis Prostatic Intraepithelial Neoplasia Prostatic Neoplasms Bone marrow-derived heparan sulfate potentiates the osteogenic activity of bone morphogenetic protein-2 (BMP-2) http://escholarship.umassmed.edu/stein/255 http://escholarship.umassmed.edu/stein/255 Thu, 26 Apr 2012 08:49:05 PDT Lowering the efficacious dose of bone morphogenetic protein-2 (BMP-2) for the repair of critical-sized bone defects is highly desirable, as supra-physiological amounts of BMP-2 have an increased risk of side effects and a greater economic burden for the healthcare system. To address this need, we explored the use of heparan sulfate (HS), a structural analog of heparin, to enhance BMP-2 activity. We demonstrate that HS isolated from a bone marrow stromal cell line (HS-5) and heparin each enhances BMP-2-induced osteogenesis in C2C12 myoblasts through increased ALP activity and osteocalcin mRNA expression. Commercially available HS variants from porcine kidney and bovine lung do not generate effects as great as HS5. Heparin and HS5 influence BMP-2 activity by (i) prolonging BMP-2 half-life, (ii) reducing interactions between BMP-2 with its antagonist noggin, and (iii) modulating BMP2 distribution on the cell surface. Importantly, long-term supplementation of HS5 but not heparin greatly enhances BMP-2-induced bone formation in vitro and in vivo. These results show that bone marrow-derived HS effectively supports bone formation, and suggest its applicability in bone repair by selectively facilitating the delivery and bioavailability of BMP-2.

]]> Diah S. Bramono et al. Bone Morphogenetic Protein 2 Heparitin Sulfate Epigenetic regulation of early osteogenesis and mineralized tissue formation by a HOXA10-PBX1-associated complex http://escholarship.umassmed.edu/stein/254 http://escholarship.umassmed.edu/stein/254 Thu, 26 Apr 2012 08:49:02 PDT Homeodomain-containing (HOX) factors such as the abdominal class homeodomain protein HOXA10 and the TALE-family protein PBX1 form coregulatory complexes and are potent transcriptional and epigenetic regulators of tissue morphogenesis. We have identified that HOXA10 and PBX1 are expressed in osteoprogenitors; however, their role in osteogenesis has not been established. To determine the mechanism of HOXA10-PBX-mediated regulation of osteoblast commitment and the related gene expression, PBX1 or HOX10 were depleted (shRNA or genetic deletion, respectively) or exogenously expressed in C3H10T1/2, bone marrow stromal progenitors, and MC3T3-E1 (preosteoblast) cells. Overexpression of HOXA10 increased the expression of osteoblast-related genes, osteoblast differentiation and mineralization; expression of PBX1 impaired osteogenic commitment of pluripotent cells and the differentiation of osteoblasts. In contrast, the targeted depletion of PBX1 by shRNA increased the expression of bone marker genes (osterix, alkaline phosphatase, BSP, and osteocalcin). Chromatin-associated PBX1 and HOXA10 were present at osteoblast-related gene promoters preceding gene expression, but PBX1 was absent from promoters during the transcription of bone-related genes, including osterix (Osx). Further, PBX1 complexes were associated with histone deacetylases normally linked with chromatin inactivation. Loss of PBX1 but not of HOXA10 from the Osx promoter was associated with increases in the recruitment of histone acetylases (p300), as well as decreased H3K9 methylation, reflecting transcriptional activation. We propose PBX1 plays a central role in attenuating the activity of HOXA10 as an activator of osteoblast-related genes and functions to establish the proper timing of gene expression during osteogenesis, resulting in proper matrix maturation and mineral deposition in differentiated osteoblasts.

]]> Jonathan A. R. Gordon et al. Animals Bone Marrow Cells Calcification, Physiologic Cell Differentiation *Epigenesis, Genetic *Gene Expression Regulation, Developmental HEK293 Cells Homeodomain Proteins Humans Mice Multiprotein Complexes Osteoblasts Osteogenesis Promoter Regions, Genetic RNA, Small Interfering Stromal Cells Transcription Factors Functional coupling of transcription factor HiNF-P and histone H4 gene expression during pre- and post-natal mouse development http://escholarship.umassmed.edu/stein/253 http://escholarship.umassmed.edu/stein/253 Thu, 26 Apr 2012 08:48:59 PDT Transcription factor Histone Nuclear Factor P (HiNF-P; gene symbol Hinfp) mediates cell cycle control of histone H4 gene expression to support the packaging of newly replicated DNA as chromatin. The HiNF-P/p220(NPAT) complex controls multiple H4 genes in established human cell lines and is critical for cell proliferation. The mouse Hinfp(LacZ) null allele causes early embryonic lethality due to a blastocyst defect. However, neither Hinfp function nor its temporal expression relative to histone H4 genes during fetal development has been explored. Here, we establish that expression of Hinfp is biologically coupled with expression of twelve functional mouse H4 genes during pre- and post-natal tissue-development. Both Hinfp and H4 genes are robustly expressed at multiple embryonic (E) days (from E5.5 to E15.5), coincident with ubiquitous LacZ staining driven by the Hinfp promoter. Five highly expressed mouse H4 genes (Hist1h4d, Histh4f, Hist1h4m and Hist2h4) account for >90% of total histone H4 mRNA throughout development. Post-natal expression of H4 genes in mice is most evident in lung, spleen, thymus and intestine, and with few exceptions (e.g., adult liver) correlates with Hinfp gene expression. Histone H4 gene expression decreases butHinfp levels remain constitutive upon cell growth inhibition in culture. The in vivo co-expression of Hinfp and histone H4 genes is consistent with the biological function of Hinfp as a principal transcriptional regulator of histone H4 gene expression during mouse development.

]]> Lijun Liu et al. Animals Blastocyst Cell Cycle Cells, Cultured Embryo, Mammalian Gene Expression *Gene Expression Regulation, Developmental Histones Mice Mice, Inbred C57BL Repressor Proteins Tissue Distribution A program of microRNAs controls osteogenic lineage progression by targeting transcription factor Runx2 http://escholarship.umassmed.edu/stein/252 http://escholarship.umassmed.edu/stein/252 Thu, 26 Apr 2012 08:48:56 PDT Lineage progression in osteoblasts and chondrocytes is stringently controlled by the cell-fate-determining transcription factor Runx2. In this study, we directly addressed whether microRNAs (miRNAs) can control the osteogenic activity of Runx2 and affect osteoblast maturation. A panel of 11 Runx2-targeting miRNAs (miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-137, miR-204, miR-205, miR-217, miR-218, and miR-338) is expressed in a lineage-related pattern in mesenchymal cell types. During both osteogenic and chondrogenic differentiation, these miRNAs, in general, are inversely expressed relative to Runx2. Based on 3'UTR luciferase reporter, immunoblot, and mRNA stability assays, each miRNA directly attenuates Runx2 protein accumulation. Runx2-targeting miRNAs differentially inhibit Runx2 protein expression in osteoblasts and chondrocytes and display different efficacies. Thus, cellular context contributes to miRNA-mediated regulation of Runx2. All Runx2-targeting miRNAs (except miR-218) significantly impede osteoblast differentiation, and their effects can be reversed by the corresponding anti-miRNAs. These findings demonstrate that osteoblastogenesis is limited by an elaborate network of functionally tested miRNAs that directly target the osteogenic master regulator Runx2.

]]> Ying Zhang et al. 3' Untranslated Regions Animals Base Sequence Blotting, Western Cell Differentiation Cell Line Cell Lineage Chondrocytes Core Binding Factor Alpha 1 Subunit Gene Expression Profiling Gene Expression Regulation Luciferases Mice MicroRNAs NIH 3T3 Cells Osteoblasts Osteogenesis RNA Stability RNA, Messenger Runx2 protein expression utilizes the Runx2 P1 promoter to establish osteoprogenitor cell number for normal bone formation http://escholarship.umassmed.edu/stein/251 http://escholarship.umassmed.edu/stein/251 Thu, 26 Apr 2012 08:48:53 PDT The Runt-related transcription factor, Runx2, is essential for osteogenesis and is controlled by both distal (P1) and proximal (P2) promoters. To understand Runx2 function requires determination of the spatiotemporal activity of P1 and P2 to Runx2 protein production. We generated a mouse model in which the P1-derived transcript was replaced with a lacZ reporter allele, resulting in loss of P1-derived protein while simultaneously allowing discrimination between the activities of the two promoters. Loss of P1-driven expression causes developmental defects with cleidocranial dysplasia-like syndromes that persist in the postnatal skeleton. P1 activity is robust in preosteogenic mesenchyme and at the onset of bone formation but decreases as bone matures. Homozygous Runx2-P1(lacZ/lacZ) mice have a normal life span but exhibit severe osteopenia and compromised bone repair in adult mice because of osteoblastic defects and not increased osteoclastic resorption. Gene expression profiles of bone, immunohistochemical studies, and ex vivo differentiation using calvarial osteoblasts and marrow stromal cells identified mechanisms for the skeletal phenotype. The findings indicate that P1 promoter activity is necessary for generating a threshold level of Runx2 protein to commit sufficient osteoprogenitor numbers for normal bone formation. P1 promoter function is not compensated via the P2 promoter. However, the P2 transcript with compensatory mechanisms from bone morphogenetic protein (BMP) and Wnt signaling is adequate for mineralization of the bone tissue that does form. We conclude that selective utilization of the P1 and P2 promoters enables the precise spatiotemporal expression of Runx2 necessary for normal skeletogenesis and the maintenance of bone mass in the adult.

]]> Julie C. Liu et al. Animals Bone Diseases, Developmental Bone Diseases, Metabolic Bone Regeneration Calcification, Physiologic Core Binding Factor Alpha 1 Subunit Gene Expression Regulation Mice Mice, Transgenic Osteoblasts Osteogenesis Promoter Regions, Genetic Stem Cells Identification of RUNX3 as a component of the MST/Hpo signaling pathway http://escholarship.umassmed.edu/stein/250 http://escholarship.umassmed.edu/stein/250 Thu, 26 Apr 2012 08:48:50 PDT Recent genetic screens of fly mutants and molecular analysis have revealed that the Hippo (Hpo) pathway controls both cell proliferation and cell death. Deregulation of its human counterpart (the MST pathway) has been implicated in human cancers. However, how this pathway is linked with the known tumor suppressor network remains to be established. RUNX3 functions as a tumor suppressor of gastric cancer, lung cancer, bladder cancer, and colon cancer. Here, we show that RUNX3 is a principal and evolutionarily conserved component of the MST pathway. SAV1/WW45 facilitates the close association between MST2 and RUNX3. MST2, in turn, stimulates the SAV1-RUNX3 interaction. In addition, we show that siRNA-mediated RUNX3 knockdown abolishes MST/Hpo-mediated cell death. By establishing that RUNX3 is an endpoint effector of the MST pathway and that RUNX3 is capable of inducing cell death in cooperation with MST and SAV1, we define an evolutionarily conserved novel regulatory mechanism loop for tumor suppression in human cancers.

]]> Boram Min et al. Animals Biological Evolution Cell Cycle Proteins Cell Death Core Binding Factor Alpha 3 Subunit Drosophila Drosophila Proteins Gene Expression Regulation HEK293 Cells Humans Intracellular Signaling Peptides and Proteins MAP Kinase Kinase Kinases Models, Molecular Mutation Protein Binding Protein Conformation Protein-Serine-Threonine Kinases Signal Transduction Two-Hybrid System Techniques Role of the WWOX tumor suppressor gene in bone homeostasis and the pathogenesis of osteosarcoma http://escholarship.umassmed.edu/stein/249 http://escholarship.umassmed.edu/stein/249 Thu, 26 Apr 2012 08:48:47 PDT Osteosarcoma is the most common primary bone malignancy in children with unknown etiology and often with poor clinical outcome. In recent years, a critical role has emerged for the WW domain-containing oxidoreductase (WWOX) in osteosarcoma and bone biology. WWOX is a tumor suppressor that is deleted or attenuated in most human tumors. Wwox-deficient mice develop osteosarcoma and a bone metabolic disease characterized by hypocalcemia and osteopenia. Studies of human osteosarcomas have revealed that the WWOX gene is deleted in 30% of cases and WWOX protein is absent or reduced in approximately 60% of tumors. Further, WWOX levels are attenuated in the majority of osteosarcoma cells, in which ectopic expression is associated with reduced proliferation, migration, invasion and tumorigenicity. At the molecular level, WWOX associates with RUNX2 and suppresses its transcriptional activity in osteoblasts and in cancer cells. This review provides new insights on the current knowledge of the spectrum of WWOX activities and future directions for the role of WWOX in bone biology and osteosarcoma.

]]> Sara Del Mare et al. Oxidoreductases Tumor Suppressor Proteins TRAP-1, the mitochondrial Hsp90 http://escholarship.umassmed.edu/stein/248 http://escholarship.umassmed.edu/stein/248 Thu, 26 Apr 2012 08:48:44 PDT Protein folding quality control does not occur randomly in cells, but requires the action of specialized molecular chaperones compartmentalized in subcellular microenvironments and organelles. Fresh experimental evidence has now linked a mitochondrial-specific Heat Shock Protein-90 (Hsp90) homolog, Tumor Necrosis Factor Receptor-Associated Protein-1 (TRAP-1) to pleiotropic signaling circuitries of organelle integrity and cellular homeostasis. TRAP-1-directed compartmentalized protein folding is broadly exploited in cancer and neurodegenerative diseases, presenting new opportunities for therapeutic intervention in humans. This article is part of a Special Issue entitled: Heat Shock Protein 90 (Hsp90).

]]> Dario C. Altieri et al. HSP90 Heat-Shock Proteins Heparan Sulfate Enhances the Self-Renewal and Therapeutic Potential of Mesenchymal Stem Cells from Human Adult Bone Marrow http://escholarship.umassmed.edu/stein/247 http://escholarship.umassmed.edu/stein/247 Thu, 26 Apr 2012 08:48:40 PDT Insufficient cell number hampers therapies utilizing adult human mesenchymal stem cells (hMSCs) and current ex vivo expansion strategies lead to a loss of multipotentiality. Here we show that supplementation with an embryonic form of heparan sulfate (HS-2) can both increase the initial recovery of hMSCs from bone marrow aspirates and increase their ex vivo expansion by up to 13-fold. HS-2 acts to amplify a subpopulation of hMSCs harboring longer telomeres and increased expression of the MSC surface marker stromal precursor antigen-1. Gene expression profiling revealed that hMSCs cultured in HS-2 possess a distinct signature that reflects their enhanced multipotentiality and improved bone-forming ability when transplanted into critical-sized bone defects. Thus, HS-2 offers a novel means for decreasing the expansion time necessary for obtaining therapeutic numbers of multipotent hMSCs without the addition of exogenous growth factors that compromise stem cell fate.

]]> Torben Helledie et al. Mesenchymal Stem Cells Heparitin Sulfate Resolution of inflammation induces osteoblast function and regulates the Wnt signaling pathway http://escholarship.umassmed.edu/stein/246 http://escholarship.umassmed.edu/stein/246 Thu, 26 Apr 2012 08:48:37 PDT OBJECTIVE: Inflammation in the bone microenvironment stimulates osteoclast differentiation, resulting in uncoupling of resorption and formation. Mechanisms contributing to the inhibition of osteoblast function in inflammatory diseases, however, have not been elucidated. Rheumatoid arthritis (RA) is a prototype of an inflammatory arthritis that results in focal loss of articular bone. The paucity of bone repair in inflammatory diseases such as RA raises compelling questions regarding the impact of inflammation on bone formation.

METHODS: To establish the mechanisms by which inflammation regulates osteoblast activity, we characterized an innovative variant of a murine arthritis model in which inflammation is induced in C57BL/6J mice by transfer of arthritogenic K/BxN serum and allowed to resolve.

RESULTS: In the setting of resolving inflammation, bone resorption ceases and appositional, osteoblast-mediated bone formation is induced, resulting in repair of eroded bone. Resolution of inflammation is accompanied by striking changes in expression of regulators of the Wnt/beta-catenin pathway, a pathway critical for osteoblast differentiation and function. Downregulation of the Wnt antagonists sFRP1 and sFRP2 during the resolution phase parallels induction of the anabolic and pro-matrix mineralization factors Wnt10b and DKK2, demonstrating the role of inflammation in regulating Wnt signaling.

CONCLUSION: Repair of articular bone erosion occurs in the setting of resolving inflammation, accompanied by alterations in the Wnt signaling pathway. These data imply that in inflammatory diseases that result in persistent articular bone loss, strict control of inflammation may not be achieved, and may be essential for the generation of an anabolic microenvironment that supports bone formation and repair.

]]> Melissa M. Matzelle et al. Wnt Signaling Pathway Inflammation Osteoblasts Bone Resorption