Pregabalin, dosed BID, reduced neuropathic pain associated with PHN and was well tolerated. It also reduced the extent to which pain interfered with sleep. Pregabalin's effects were seen as early as week 1 and were sustained throughout the 13-week study.

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 ␣1(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 Cys 2 His 2 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. (J Bone Miner Res 2001;16:10 -23)

The renal reabsorption of glucose is mediated by two major classes of transporters. Initially, luminal glucose is concentrated in tubules by Na(+)-glucose cotransporters (Na(+)-GLUT). Afterwards, glucose reaches the blood space through facilitative glucose transporters, low-Michaelis constant (Km) GLUT1 and high-Km GLUT2. Hence, the transtubular flux of glucose could be impaired in hyperglycemia because the outwardly directed glucose gradient, from tubule to blood, is potentially lowered. However, in diabetic rats, transtubular glucose flux is not reduced but increased. In this work the molecular mechanism underlying this adaptation was examined. We tested the hypothesis that upregulation of renal tubular high-Km GLUT2 gene may compensate for the decrease in the tubule to blood glucose gradient. In rat tubules, GLUT1 protein and mRNA steady-state levels were reduced, and GLUT2 protein and mRNA levels were increased in rats after 2, 3, and 4 wk of uncontrolled streptozotocin-induced diabetes. These molecular adaptations were associated with augmented facilitative glucose flux. In summary, changes in GLUT1 and GLUT2 gene expression are important to the preservation of renal glucose reabsorption in hyperglycemia.

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. (J Bone Miner Res 1998;13:155-167) THE CONNECTION BETWEEN THE SHAPE OF A BONE CELL AND ITS GENETIC PROGRAMT HE COUPLING OF CELL SHAPE and gene expression through the linkage of dynamic skeletal networks provides a molecular framework for sensing and responding to mechanical stimuli, fluid flow, and diffusion-based chemical signaling pathways that induce changes in cell morphology.(1-10) This is particularly relevant to osteoblast gene expression. That the shape of a cell is somehow related to what that cell does has been a venerable article of faith in pathology. However, data to support the concept that molecular mechanisms link cell structure to gene expression are only recently emerging from studies in cell and molecular biology.(1-15) In the tensegrity paradigm, a subcellular scaffolding consisting of proteins from the extracellular matrix (ECM), the integrin receptors, and the cyto-and nucleoskeletons, is physically linked or "hard-wired" to the genes. (16 -18) An outstanding question of this paradigm is how a mechanical tug on the DNA contributes to the regulation of

LIM homeodomain transcription factors regulate development in complex organisms. To characterize the molecular signals required for the nuclear localization of these proteins, we examined the Lhx3 factor. Lhx3 is essential for pituitary organogenesis and motor neuron specification. By using functional fluorescent derivatives, we demonstrate that Lhx3 is found in both the nucleoplasm and nuclear matrix. Three nuclear localization signals were mapped within the homeodomain, and one was located in the carboxyl terminus.

The mechanisms underlying the coupling of type I collagen and matrix metalloproteinase (MMP) expression to cell structure and adhesion are poorly understood. We propose that nuclear matrix architectural transcription factors link cell structure and transcription via their association with nuclear matrix subdomains and by their capacity for altering promoter geometry. NP/NMP4 are nuclear matrix proteins that contain from five to eight Cys(2)His(2) zinc fingers. Some NP/NMP4 isoforms bind to the rat type I collagen alpha1(I) polypeptide chain promoter in the manner of architectural transcription factors and alter basal transcription in osteoblast-like cells (Thunyakitpisal et al. in review). Certain isoforms of NP/NMP4 are identical to CIZ, Cas-interacting zinc finger protein, a nucleocytoplasmic shuttling protein that associates with focal adhesions and regulates MMP expression [Nakamoto et al. (2000): Mol Cell Biol 20:1649-1658]. To better understand the role of subnuclear architecture in collagen and MMP expression, we mapped the osteoblast nuclear distribution of NP/NMP4 proteins and identified the functional motifs necessary for nuclear localization and nuclear matrix targeting. Immunofluorescence microscopy was used to determine the cellular and subnuclear distribution of native NP/NMP4 proteins and green fluorescent protein (GFP)-NP/NMP4 fusion proteins in osteoblast-like cells. All GFP-NP/NMP4 fusion proteins localized to the nucleus, but accumulated in distinct nuclear matrix subdomains. The zinc finger domain was necessary and sufficient for nuclear import and matrix targeting. We conclude that the arrangement of the NP/NMP4 zinc fingers largely determines the subnuclear location of these isoforms.

Parathyroid hormone (PTH) alters osteoblast morphology. How these changes in cell shape modify nuclear structure and ultimately gene expression is not known. Chronic exposure to rat PTH (1-34) [10 nM] attenuated the expression of 200, 190, and 160 kD proteins in the nuclear matrix-intermediate filament subfraction of the rat osteosarcoma cells, ROS 17/2.8 [Bidwell et al. (1994b): Endocrinology 134:1738-1744]. Here, we determined that these same PTH-responsive proteins were expressed in rat metaphyseal osteoblasts. We identified the 200 kD protein as a non-muscle myosin. Although the molecular weights, subcellular distribution, and half-lives of the 190 and 160 kD proteins were similar to topoisomerase II-alpha and -beta, nuclear matrix enzymes that mediate DNA topology, the 190 and 160 kD proteins did not interact with topoisomerase antibodies. Nevertheless, the expression of topoisomerase II-alpha, and NuMA, a component of the nuclear core filaments, was also regulated by PTH in the osteosarcoma cells. The 190 kD protein was selectively expressed in bone cells as it was not observed in OK opossum kidney cells, H4 hepatoma cells, or NIH3T3 cells. PTH attenuated mRNA expression of the PTH receptor in our cell preparations. These results demonstrate that PTH selectively alters the expression of osteoblast membrane, cytoskeletal, and nucleoskeletal proteins. Topoisomerase II-alpha, NuMA, and the 190 and 160 kD proteins may direct the nuclear PTH signalling pathways to the target genes and play a structural role in osteoblast gene expression.

No abstract