Biomedical subjects

Joseph P Bidwell

Publications and source records attributed to Joseph P Bidwell.

8 recordsLinked to original sources

PubMed2006

Identification in human osteoarthritic chondrocytes of proteins binding to the novel regulatory site AGRE in the human matrix metalloprotease 13 proximal promoter.

OBJECTIVE: Matrix metalloprotease 13 (MMP-13) plays a major role in osteoarthritic (OA) processes. We previously identified the AG-rich element (AGRE) regulatory site (GAAAAGAAAAAG) in the proximal promoter of this gene. Electrophoretic mobility shift assays (EMSAs) done with nuclear extracts from OA chondrocytes showed the presence of 2 AGRE protein-binding complexes, the formation of which depended on the pathophysiologic state (high or low) of the cells; the low OA (L-OA) chondrocytes have low MMP-13 basal levels and high interleukin-1beta (IL-1beta) inducibility, and the high OA (H-OA) chondrocytes have high MMP-13 basal levels and low IL-1beta inducibility. In this study, we sought to determine the importance of individual AGRE bases in promoter activity and to identify AGRE binding proteins from L-OA and H-OA chondrocyte complexes. METHODS: Promoter activity was determined following transient transfection into human OA chondrocytes. AGRE binding proteins were identified by mass spectroscopy. RESULTS: Individual mutations of the AGRE site differentially modulated promoter activity, indicating that the intact AGRE site is required for optimal MMP-13 expression. Damage-specific DNA binding protein 1 (DDB-1) was identified in the L-OA chondrocyte-binding complex. EMSA experiments performed with the mutation of the left AGRE site (GTGCTGAAAAAG) and nuclear extracts of L-OA chondrocytes reproduced the pattern seen in the H-OA chondrocytes. Mass spectroscopy identified p130cas as one of the proteins in this complex. Supershift experiments showed the presence of p130cas and nuclear matrix transcription factor 4 (NMP-4) in the wild-type AGRE/H-OA chondrocyte complex. CONCLUSION: These data suggest that the binding of p130(cas) and NMP-4 to the AGRE site regulates MMP-13 expression and may trigger the change in human chondrocytes from the L-OA state to the H-OA state.

Base Sequence↗

PubMed2006

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-kappaB ligand (RANKL)/osteoprotegerin (OPG)/receptor activator of NF-kappaB (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.

Alkaline Phosphatase↗

PubMed2005

Two promoters control the mouse Nmp4/CIZ transcription factor gene.

Nmp4/CIZ proteins (nuclear matrix protein 4/cas interacting zinc finger protein) contribute to gene regulation in bone, blood, and testis. In osteoblasts, they govern the magnitude of gene response to osteotropic factors like parathyroid hormone (PTH). Nmp4/CIZ is recurrently involved in acute leukemia and it has been implicated in spermatogenesis. However, these conserved proteins, derived from a single gene, are expressed in numerous tissues indicative of a more generalized housekeeping function in addition to their tissue-specific roles. To address how Nmp4/CIZ expression is governed, we characterized the 5' regulatory region of the mouse Nmp4 gene, located on chromosome 6. Two adjacent promoters P(1) [-2521 nucleotide (nt)/-597 nt] and P(2) (-2521 nt/+1 nt) initiate transcription of alternative first exons (U(1) and U(2)). Both promoters lack TATA and CCAAT boxes but contain initiator sites and CpG islands. Northern analysis revealed expression of both U(1) and U(2) in numerous adult tissues consistent with the constitutive and ubiquitous activity of a housekeeping gene. Sequence analysis identified numerous potential transcription factor-binding sites significant to osteogenesis, hematopoeisis, and gonadal development. The promoters are active in both osteoblast-like cells and in the M12 B-lymphocyte cell line. Low doses of PTH attenuated P(1)/P(2) activity in osteoblast-like cells. The Nmp4/CIZ promoters are autoregulated and deletion analysis identified regions that drive P(1) and P(2) basal activities as well as regions that contain positive and negative regulatory elements affecting transcription. The Nmp4/CIZ promoters comprise a genomic regulatory architecture that supports constitutive expression as well as cell- and tissue-specific regulation.

Animals↗

PubMed2004

Conserved amino acid sequences confer nuclear localization upon the Prophet of Pit-1 pituitary transcription factor protein.

Prophet of Pit-1 (PROP1) is a homeodomain transcription factor essential for development of the mammalian anterior pituitary gland. Studies of human patients and animal models with mutations in their Prop1 genes have established that PROP1 is required for the correct development or sustained function of the hormone-secreting cells that regulate physiological pathways controlling growth, reproduction, metabolism, and the stress response. By comparative analysis of mammalian Prop1 genes and their encoded proteins, including cloning the ovine Prop1 gene and its products, we demonstrate that two conserved basic regions (B1 and B2) of the PROP1 protein located within the homeodomain are required for nuclear localization, DNA binding, and target gene activation. Interestingly, missense mutations in the human Prop1 gene causing amino acid changes in both the B1 and B2 regions have been associated with combined pituitary hormone deficiency (CPHD) diseases, suggesting that disruption of nuclear localization may be part of the molecular basis of such diseases. The ovine Prop1 gene has three exons and two introns, a different structure compared with that of the bovine gene. Two alleles of the ovine gene were found to encode protein products with different carboxyl terminal domain sequences. We demonstrate that the two alleles are distributed in different breeds of sheep. Finally, we show for the first time that the PROP1 protein is associated with the nuclear matrix.

Alleles↗

PubMed2004

Nmp4/CIZ regulation of matrix metalloproteinase 13 (MMP-13) response to parathyroid hormone in osteoblasts.

Parathyroid hormone (PTH) regulation of matrix metalloproteinase-13 (MMP-13) expression in osteoblasts contributes to normal bone turnover. The PTH response region of the rat MMP-13 gene spans nucleotides (nt) -148 to -38 and supports binding of numerous transcription factors, including Runx2, necessary for osteoblast differentiation, c-Fos/c-Jun, and Ets-1. These trans-acting proteins mediate hormone induction via incompletely defined combinatorial interactions. Within this region, adjacent to the distal Runx2 site, is a homopolymeric(dA:dT) element (-119/-110 nt) that conforms to the consensus site for the novel transcription factor nuclear matrix protein-4/cas interacting zinc finger protein (Nmp4/CIZ). This protein regulates bone cell expression of type I collagen and suppresses BMP2-enhanced osteoblast differentiation. The aim of this study was to determine whether Nmp4/CIZ contributes to MMP-13 basal transcription and PTH responsiveness in osteoblasts. Electrophoretic mobility shift analysis confirms Nmp4/CIZ binding within the MMP-13 PTH response region. Mutation of the Nmp4/CIZ element decreases basal activity of an MMP-13 promoter-reporter construct containing the first 1329 nt of the 5'-regulatory region, and overexpression of Nmp4/CIZ protein enhances the activity of the wild-type promoter. The same mutation of the homopolymeric(dA:dT) element enhances the MMP-13 response to PTH and PGE(2). Overexpression of Nmp4/CIZ diminishes hormone induction. Mutation of both the homopolymeric(dA:dT) element and the adjacent Runx2 site further augments the PTH response. On the basis of these data and previous studies, we propose that Nmp4/CIZ is a component of a multiprotein assemblage or enhanceosome within the MMP-13 PTH response region and that, within this context, Nmp4/CIZ promotes both basal expression and hormonal synergy.

3T3 Cells↗

PubMed2003

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.

Animals↗

PubMed2003

A model for mechanotransduction in bone cells: the load-bearing mechanosomes.

The skeleton's response to mechanical force, or load, has significance to space travel, the treatment of osteoporosis, and orthodontic appliances. How bone senses and processes load remains largely unknown. The cellular basis of mechanotransduction, however, likely involves the integration of diffusion-controlled signaling pathways with a solid-state scaffold linking the cell membrane to the genes. Here, we integrate various concepts from models of connective membrane skeleton proteins, cellular tensegrity, and nuclear matrix architectural transcription factors, to describe how a load-induced deformation of bone activates a change in the skeletal genetic program. We propose that mechanical information is relayed from the bone to the gene in part by a succession of deformations, changes in conformations, and translocations. The load-induced deformation of bone is converted into the deformation of the sensor cell membrane. This, in turn, drives conformational changes in membrane proteins of which some are linked to a solid-state signaling scaffold that releases protein complexes capable of carrying mechanical information, "mechanosomes", into the nucleus. These mechanosomes translate this information into changes in the geometry of the 5' regulatory region of target gene DNA altering gene activity; bending bone ultimately bends genes. We identify specific candidate proteins fitting the profile of load-signaling mechanosomes.

Animals↗

PubMed2002

DNA binding and gene activation properties of the Nmp4 nuclear matrix transcription factors.

Splice variants of the Nmp4 gene include nuclear matrix transcription factors that regulate the type I collagen alpha1(I) polypeptide chain (COL1A1) promoter and several matrix metalloproteinase (MMP) genes. To date, these are the only Cys(2)His(2) zinc finger proteins known to bind within the minor groove of homopolymeric (dA.dT) DNA. Nmp4 isoforms contain from 5 to 8 Cys(2)His(2) zinc fingers, an SH3-binding domain that overlaps with a putative AT-hook and a polyglutamine-alanine repeat (poly(QA)). To determine the mechanistic significance of Cys(2)His(2) zinc finger association with this unusual consensus DNA binding element, we identified the Nmp4 DNA-binding and transcriptional activation domains. Zinc fingers 2, 3, and 6 mediated association with the homopolymeric (dA.dT) COL1A1/MMP DNA consensus element. The N terminus of the Nmp4 protein exhibited a strong trans-activation capacity when fused to the GAL4 DNA-binding domain, but this activity was masked within the context of the full-length Nmp4-GAL4 DNA-binding domain chimera. However, upon binding to the COL1A1/MMP homopolymeric (dA.dT) element, the native Nmp4 protein up-regulated transcription, and the poly(QA) domain acquired a significant role in trans-activation. We propose that allosteric effects induced upon zinc finger association with the homopolymeric (dA.dT) minor groove confer context-specific functionality to this unusual family of Cys(2)His(2) transcription factors.

Antigens, Nuclear↗