Marta B. Alvarez

Fluid Shear Stress Induces β-Catenin Signaling in Osteoblasts

β-Catenin plays a dual role in cells: one at cell–cell junctions and one regulating gene transcription together with TCF (T-cell Factor) in the nucleus. Recently, a role for β-catenin in osteoblast differentiation and gene expression has begun to be elucidated. Herein we investigated the effects of fluid shear stress (FSS) on β-catenin signaling. FSS is a well-characterized anabolic stimulus for osteoblasts; however, the molecular mechanisms for the effects of this stimulation remain largely unknown. We found that 1 hour of laminar FSS (10 dynes/cm2) induced translocation of β-catenin to the nucleus and activated a TCF-reporter gene. Analysis of upstream signals that may regulate β-catenin signaling activity revealed two potential mechanisms for increased β-catenin signaling. First, FSS induced a transient, but significant, increase in the phosphorylation of both glycogen synthase kinase 3β (GSK-3β) and Akt. Second, FSS reduced the levels of β-catenin associated with N-cadherin, suggesting that less sequestration of β-catenin by cadherins occurs in osteoblasts subjected to FSS. Functional analysts of potential genes regulated by β-catenin signaling in osteoblasts revealed two novel observations. First, endogenous, nuclear β-catenin purified from osteoblasts formed a complex with a TCF -binding element in the cyclooxygenase-2 promoter, and, second, overexpression of either a constitutively active β-catenin molecule or inhibition of GSK-3β activity increased basal cyclooxygenase-2 levels. Together, these data demonstrate for the first time that FSS modulates the activity of both GSK-3β and β-catenin and that these signaling molecules regulate cyclooxygenase-2 expression in osteoblasts.

Nuclear Matrix Proteins and Osteoblast Gene Expression

The molecular mechanisms that couple osteoblast structure and gene expression are emerging from recent studies on the bone extracellular matrix, integrins, the cytoskeleton, and the nucleoskeleton (nuclear matrix). These proteins form a dynamic structural network, the tissue matrix, that physically links the genes with the substructure of the cell and its substrate. The molecular analog of cell structure is the geometry of the promoter. The degree of supercoiling and bending of promoter DNA can regulate transcriptional activity. Nuclear matrix proteins may render a change in cytoskeletal organization into a bend or twist in the promoter of target genes. We review the role of nuclear matrix proteins in the regulation of gene expression with special emphasis on osseous tissue. Nuclear matrix proteins bind to the osteocalcin and type I collagen promoters in osteoblasts. One such protein is Cbfa1, a recently described transcriptional activator of osteoblast differentiation. Although their mechanisms of action are unknown, some nuclear matrix proteins may act as “architectural” transcription factors, regulating gene expression by bending the promoter and altering the interactions between other trans‐acting proteins. The osteoblast nuclear matrix is comprised of cell‐ and phenotype‐specific proteins including proteins common to all cells. Nuclear matrix proteins specific to the osteoblast developmental stage and proteins that distinguish osteosarcoma from the osteoblast have been identified. Recent studies indicating that nuclear matrix proteins mediate bone cell response to parathyroid hormone and vitamin D are discussed.

Context-dependent transcription: All politics is local

An organism ultimately reflects the coordinate expression of its genome. The misexpression of a gene can have catastrophic consequences for an organism, yet the mechanics of transcription is a local phenomenon within the cell nucleus. Chromosomal and nuclear position often dictate the activity of a specific gene. Transcription occurs in territories and in discrete localized foci within these territories. The proximity of a gene or trans-acting factor to heterochromatin can have profound functional significance. The organization of heterochromatin changes with cell development, thus conferring temporal changes on gene activity. The protein-protein interactions that engage the trans-acting factor also contribute to context-dependent transcription. Multi-protein assemblages known as enhanceosomes govern gene expression by local committee thus dictating regional transcription factor function. Local DNA architecture can prescribe enhancesome membership. The local bending of the double helix, typically mediated by architectural transcription factors, is often critical for stabilizing enhanceosomes formed from trans-acting proteins separated over small and large distances. The recognition element to which a transcription factor binds is of functional significance because DNA may act as an allosteric ligand influencing the conformation and thus the activity of the transactivation domain of the binding protein, as well as the recruitment of other proteins to the enhanceosome. Here, we review and attempt to integrate these local determinants of gene expression.

Cloning and functional analysis of a family of nuclear matrix transcription factors (NP/NMP4) that regulate type I collagen expression in osteoblasts

Collagen expression is coupled to cell structure in connective tissue. We propose that nuclear matrix architectural transcription factors link cell shape with collagen promoter geometry and activity. We previously indicated that nuclear matrix proteins (NP/NMP4) interact with the rat type I collagen α1(I) polypeptide chain (COL1A1) promoter at two poly(dT) sequences (sites A and B) and bend the DNA. Here, our objective was to determine whether NP/NMP4‐COL1A1 binding influences promoter activity and to clone NP/NMP4. Promoter‐reporter constructs containing 3.5 kilobases (kb) of COL1A1 5′ flanking sequence were fused to a reporter gene. Mutation of site A or site B increased promoter activity in rat UMR‐106 osteoblast‐like cells. Several full‐length complementary DNAs (cDNAs) were isolated from an expression library using site B as a probe. These clones expressed proteins with molecular weights and COL1A1 binding activity similar to NP/NMP4. Antibodies to these proteins disrupted native NP/NMP4‐COL1A1 binding activity. Overexpression of specific clones in UMR‐106 cells repressed COL1A1 promoter activity. The isolated cDNAs encode isoforms of Cys2His2 zinc finger proteins that contain an AT‐hook, a motif found in architectural transcription factors. Some of these isoforms recently have been identified as Cas‐interacting zinc finger proteins (CIZ) that localize to fibroblast focal adhesions and enhance metalloproteinase gene expression. We observed NP/NMP4/CIZ expression in osteocytes, osteoblasts, and chondrocytes in rat bone. We conclude that NP/NMP4/CIZ is a novel family of nuclear matrix transcription factors that may be part of a general mechanical pathway that couples cell structure and function during extracellular matrix remodeling.

HMGB1 expression and release by bone cells

Immune and bone cells are functionally coupled by pro‐inflammatory cytokine intercellular signaling networks common to both tissues and their crosstalk may contribute to the etiologies of some immune‐associated bone pathologies. For example, the receptor activator of NF‐κB ligand (RANKL)/osteoprotegerin (OPG)/receptor activator of NF‐κB (RANK) signaling axis plays a critical role in dendritic cell (DC) function as well as bone remodeling. The expression of RANKL by immune cells may contribute to bone loss in periodontitis, arthritis, and multiple myeloma. A recent discovery reveals that DCs release the chromatin protein high mobility group box 1 (HMGB1) as a potent immunomodulatory cytokine mediating the interaction between DCs and T‐cells, via HMGB1 binding to the membrane receptor for advanced glycation end products (RAGE). To determine whether osteoblasts or osteoclasts express and/or release HMGB1 into the bone microenvironment, we analyzed tissue, cells, and culture media for the presence of this molecule. Our immunohistochemical and immunocytochemical analyses demonstrate HMGB1 expression in primary osteoblasts and osteoclasts and that both cells express RAGE. HMGB1 is recoverable in the media of primary osteoblast cultures and cultures of isolated osteoclast precursors and osteoclasts. Parathyroid hormone (PTH), a regulator of bone remodeling, attenuates HMGB1 release in cultures of primary osteoblasts and MC3T3‐E1 osteoblast‐like cells but augments this release in the rat osteosarcoma cell line UMR 106‐01, both responses primarily via activation of adenylyl cyclase. PTH‐induced HMGB1 discharge by UMR cells exhibits similar release kinetics as reported for activated macrophages. These data confirm the presence of the HMGB1/RAGE signaling axis in bone. J. Cell. Physiol. 207: 480–490, 2006.

Rat Osteoblast and Osteosarcoma Nuclear Matrix Proteins Bind with Sequence Specificity to the Rat Type I Collagen Promoter

The nuclear matrix mediates the 3-dimensional organization of DNA and supports DNA replication and its transcription. We hypothesize that the osteoblast nuclear matrix contributes to the transcriptional control of type I collagen (COL1A1) expression. Cis-regulatory elements of the rat COL1A1 promoter that control osteoblast expression in vivo are between −2.3 and −1.67 kilobase pairs (kb) but lie within −3.5 and −2.3 kb in cultured bone cells. This may result from differences in cell architecture between osteoblasts in tissue and those in vitro. Our aim was to identify osteoblast nuclear matrix proteins (NMPs) that associated with sequence-specificity to the COL1A1 promoter. We used osteoblasts from the rat metaphyseal femur and the rat osteosarcoma cells, ROS 17/2.8. Nuclear matrix and soluble nuclear proteins were obtained as separate subfractions. Gel mobility shift analysis, using fragments of the COL1A1 promoter, was used to identify DNA-binding proteins in the nuclear subfractions. A NMP-DNA interac...

PTH-responsive osteoblast nuclear matrix architectural transcription factor binds to the rat type I collagen promoter.

In connective tissue, cell structure contributes to type I collagen expression. Differences in osteoblast microarchitecture may account for the two distinct cis elements regulating basal expression, in vivo and in vitro, of the rat type I collagen α1(I) polypeptide chain (COL1A1). The COL1A1 promoter conformation may be the penultimate culmination of osteoblast structure. Architectural transcription factors bind to the minor groove of AT‐rich DNA and bend it, altering interactions between other trans‐acting proteins. Similarly, nuclear matrix (NM) proteins bind to the minor groove of AT‐rich matrix‐attachment regions, regulating transcription by altering DNA structure. We propose that osteoblast NM architectural transcription factors link cell structure to promoter geometry and COL1A1 transcription. Our objective was to identify potential osteoblast NM architectural transcription factors near the in vitro and in vivo regulatory regions of the rat COL1A1 promoter. Nuclear protein‐promoter interactions were analyzed by gel shift analysis and related techniques. NM extracts were derived from rat osteosarcoma cells and from rat bone. The NM protein, NMP4, and a soluble nuclear protein, NP, both bound to two homologous poly(dT) elements within the COL1A1 in vitro regulatory region and proximal to the in vivo regulatory element. These proteins bound within the minor groove and bent the DNA. Parathyroid hormone increased NP/NMP4 binding to both poly(dT) elements and decreased COL1A1 mRNA in the osteosarcoma cells. NP/NMP4‐COL1A1 promoter interactions may represent a molecular pathway by which osteoblast structure is coupled to COL1A1 expression. J. Cell. Biochem. 69:336–352.

Functional Impairment of Bone Formation in the Pathogenesis of Osteoporosis: The Bone Marrow Regenerative Competence

The skeleton is a high-renewal organ that undergoes ongoing cycles of remodeling. The regenerative bone formation arm ultimately declines in the aging, postmenopausal skeleton, but current therapies do not adequately address this deficit. Bone marrow is the primary source of the skeletal anabolic response and the mesenchymal stem cells (MSCs), which give rise to bone matrix-producing osteoblasts. The identity of these stem cells is emerging, but it now appears that the term ‘MSC’ has often been misapplied to the bone marrow stromal cell (BMSC), a progeny of the MSC. Nevertheless, the changes in BMSC phenotype associated with age and estrogen depletion likely contribute to the attenuated regenerative competence of the marrow and may reflect alterations in MSC phenotype. Here we summarize current concepts in bone marrow MSC identity, and within this context, review recent observations on changes in bone marrow population dynamics associated with aging and menopause.

Nmp4/CIZ contributes to fluid shear stress induced MMP-13 gene induction in osteoblasts

The expression of matrix metalloproteinase‐13 (MMP‐13), involved in bone turnover, is elevated in stretched MC3T3‐E1 osteoblast‐like cells. Strain‐mediated forces impact bone remodeling due in large part to the movement of fluid through the canalicular‐lacunar network. The resulting fluid shear stress (FSS) over the surface membranes of bone cells initiates bone remodeling. Although the nuclear events mediating putative FSS‐induced changes in osteoblast MMP‐13 transcription are unknown, previous studies with bone cells suggest an overlap between osteoblast FSS‐ and PTH‐induced signal response pathways. MMP‐13 PTH response is regulated by a 110 bp 5′ regulatory region, conserved across the mouse, rat, and human genes, that supports the binding of numerous transcription factors including Runx2, c‐fos/c‐jun, Ets‐1, and nuclear matrix protein 4/cas interacting zinc finger protein (Nmp4/CIZ) a nucleocytoplasmic shuttling trans‐acting protein that attenuates PTH‐driven transcription. Nmp4/CIZ also binds p130cas, an adaptor protein implicated in mechanotransduction. Here we sought to determine whether Nmp4/CIZ contributes to FSS‐induced changes in MMP‐13 transcription. FSS (12 dynes/cm2, 3–5 h) increased MMP‐13 promoter‐reporter activity approximately two‐fold in MC3T3‐E1 osteoblast‐like cells attended by a comparable increase in mRNA expression. This was accompanied by a decrease in Nmp4/CIZ binding to its cis‐element within the PTH response region, the mutation of which abrogated the MMP‐13 response to FSS. Interestingly, FSS enhanced Nmp4/CIZ promoter activity and induced p130cas nuclear translocation. We conclude that the PTH regulatory region of MMP‐13 also contributes to FSS response and that Nmp4/CIZ plays similar but distinct roles in mediating hormone‐ and FSS‐driven induction of MMP‐13 in bone cells. J. Cell. Biochem. 102: 1202–1213, 2007.

Immortalization and characterization of osteoblast cell lines generated from wild-type and Nmp4-null mouse bone marrow stromal cells using murine telomerase reverse transcriptase (mTERT).

Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH‐induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4‐knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH‐responsiveness permitted further studies with single WT and KO clones. We recently demonstrated that PTH‐induced c‐fos femoral mRNA expression is enhanced in Nmp4‐KO mice and in the present study we observed that hormone stimulated either an equivalent or modestly enhanced increase in c‐fos mRNA expression in both primary null and KO clone cells depending on PTH concentration. The null primary osteoblasts and KO clone cells exhibited a transiently enhanced response to bone morphogenetic protein 2 (BMP2). The clones exhibited lower and higher expressions of the PTH receptor (Pthr1) and the BMP2 receptor (Bmpr1a, Alk3), respectively, as compared to primary cells. These immortalized cell lines will provide a valuable tool for disentangling the complex functional roles underlying Nmp4/CIZ regulation of bone anabolism. J. Cell. Physiol. 227: 1873–1882, 2012.