Nuclear Matrix Proteins and Osteoblast Gene ... - Wiley Online Library

JOURNAL OF BONE AND MINERAL RESEARCH Volume 13, Number 2, 1998 Blackwell Science, Inc. © 1998 American Society for Bone and Mineral Research

Review Nuclear Matrix Proteins and Osteoblast Gene Expression JOSEPH P. BIDWELL,1,2 MARTA ALVAREZ,3 HILARY FEISTER,2 JUDE ONYIA,3,4 and JANET HOCK1,4

ABSTRACT 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 PROGRAM

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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

1

Department of Periodontics, Indiana University School of Dentistry, Indianapolis, Indiana, U.S.A. Department of Anatomy, Indiana University School of Medicine, Indianapolis, Indiana, U.S.A. 3 Department of Oral Biology, Indiana University School of Dentistry, Indianapolis, Indiana, U.S.A. 4 Endocrine Division, Eli Lilly & Co, Indianapolis, Indiana, U.S.A. 2

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BIDWELL ET AL. TABLE 1. PROTEINS THAT REGULATE DNA STRUCTURE

1. Architectural transcription factors HMG proteins(22,128,130–132) LEF-1(129,134,135) c-jun/c-fos(25) POU proteins(27) YY1(30) runt domain proteins(32) 2. Nuclear matrix MAR-binding proteins Topoisomerase II-a and -b(116–118,122,123) SATB1(34) bright(36,74) nucleolin(35) 3. Osteoblast nuclear matrix proteins NMP1/YY1(37,142,164) NMP2/Cbfa1(28,75,142) NMP4(138,146) topoisomerase II-a and -b(105)

High mobility group proteins. Large family of proteins that bend DNA in a variety of promoter contexts. Regulates T-cell receptor a-gene enhancer by bending DNA. fos-jun heterodimers and Jun homodimers induce flexure at the AP-1 site. Oct-1, Oct-2A, Oct-6, and Pit-1 induce DNA bending. Ubiquitously expressed protein that regulates c-fos promoter through DNA bending. These transcriptional regulators bend DNA, perhaps stabilizing higher nucleoprotein complexes. Ubiquitously expressed. May regulate cell development by mediating changes in supercoiling. Predominantly expressed in thymus suggesting a role in gene regulation. B cell–specific protein required for IgH gene transcription. Ubiquitously expressed MAR-binding protein. Localized to both nuclear matrix and chromatin of osteoblasts. Binds to osteocalcin promoter and mediates repression of vitamin D induction. Binds to osteocalcin promoter. Transcriptional activator of osteoblast differentiation. Member of the Runt family. Binds to type I collagen promoter. PTH-responsive and bends DNA. Differentially expressed in immature and mature bone cells. Topo II-a expression is responsive to PTH.

promoter activity, i.e., what are the mechanisms that underlie the cytoskeletal-nuclear communication pathway?(18 –20) Recent studies on transcription indicate that promoter geometry, i.e., the degree of supercoiling and/or the amplitude of a directed bend or loop in the DNA, plays a significant role in regulating the activity of some genes.(21– 35) Architectural transcription factors mediate this promoter structure by inducing bends or twists in the DNA.(21– 36) Some of these architectural transcription factors have been localized to the nucleoskeleton or nuclear matrix,(30,32,34 –38) thus physically linking the cell substructure with promoter geometry and gene activity (Table 1). This model suggests that a change in the genetic program of the cell is more than the result of a redistribution of phosphate groups among proteins but includes a coordinated realignment of structural elements in the cell and nucleus, including the target gene promoter.(16 –18) Here, we review the role of nuclear matrix proteins in gene expression with particular emphasis on the osteoblast. The nuclear matrix will be described as an integral component of the cell’s substructure, linking the geometry of the gene with the periphery of the cell.

THE TISSUE MATRIX PHYSICALLY LINKS THE GENES WITH THE CELL SUBSTRUCTURE AND THE ECM The tissue matrix is the linkage between the structural networks of the ECM, the cytoplasm (cytoskeleton), and

the nucleus (nucleoskeleton or nuclear matrix), that link DNA to the cell periphery (Fig. 1).(39 – 42) Fibronectin, laminin, and the collagens of the ECM, the cell– cell and cell– substrate receptors (e.g., the integrins), the microfilaments, microtubules, and intermediate filaments, the lamins, and the myriad of fibrillogranular proteins of the nuclear matrix all contribute to this tissue–matrix complex.(39,41,42) Tissue matrix proteins play a vital role in cell signaling and underlie the application of the tensegrity theory to signal transduction.(43– 45) Tensegrity is defined as a structural system of discontinuous compression elements coupled by continuous tension cables forming a dynamic structure.(43– 45) Although a detailed discussion is beyond the scope of this review, the consideration of the cell in the context of an interacting tension and compression system confers a capacity for vectorial chemomechanical coupling to second messenger mobilization.(43– 45) In this paradigm, oscillating second messenger signals may propagate and pass amplitude- and frequency-dependent information along structural elements.(43– 45) Protein kinase CK2, a ubiquitously expressed protein putatively involved in growth control, associates with the nuclear matrix, thus integrating soluble and “solid-state” signaling pathways.(44) Conversely, the soluble signaling pathways can effect biochemical modifications, polymerization or depolymerization, cross-linking, and contractile and vibratory movements of the skeletal networks.(39) Does the tissue matrix control gene expression or does gene expression regulate the organization of the tissue ma-

OSTEOBLAST NUCLEAR MATRIX

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FIG. 1. Osteoblast tissue matrix. The cell substructure is comprised of integrated structural networks of the extracellular matrix, the cytosol, and the nucleus.

trix? Recent evidence indicates a reciprocal relationship. Chen et al.(2) demonstrated that cell shape determined whether individual cells divided or died. Using microfabricated, nonadhesive surfaces coated with fibronectin in various geometric patterns, it was determined that human and bovine capillary endothelial cells switched from growth to apoptosis as the total cell–matrix contact area decreased. Additionally, while maintaining the total cell–matrix contact area constant but changing the degree of cell spreading by altering the spacing between multiple focal adhesionsized islands, cell growth increased and apoptosis decreased with an increase in projected cell area.(2) Similarly, a recent study has demonstrated that depolymerization of microtubules activates the transcription factor NF-kB and induces NF-kB– dependent gene expression in HeLa cells.(20) Colchicine, a disrupter of microtubules, activates mitogen-activated protein (MAP) kinase(46) and protein kinase A (PKA)(47) in rat 3Y1 cells and human monocytes, respectively. The recent characterization of the novel protein kinases misshapen and RON, a receptor protein kinase, is evidence that gene expression can, in turn, regulate cell shape.(48,49) The misshapen gene is required for the normal shape and orientation of Drosophila photoreceptor cells.(48) This protein is also expressed in the embryonic mesoderm, pole plasm, and other sites of cell shape change and may be part of a signal transduction pathway that mediates cytoskeletal organization. The activation of RON alters macrophage morphology and induces migra-

tion of macrophages and epithelial cells.(49) The signal transduction pathway that mediates this change in cell shape includes the binding of macrophage stimulating protein to RON, inducing a phosphorylation of the receptor and phosphatidylinositol-3 kinase (PI-3 kinase); the phosphorylated RON forms a complex with the PI-3 kinase critical to mediating the shape change.(49)

Osteoblast tissue matrix proteins The components of the osteoblast tissue matrix play a significant role in the regulation of gene expression.(50 –54) The interaction between the a2b1 integrin receptor with matrix type I collagen was determined to be critical for mediating osteoblastic differentiation in the bone cell line MC3T3-E1.(52) The progressive development of MC3T3-E1 cells toward the mature osteoblast phenotype includes an increase in the expression of alkaline phosphatase (ALP) and a decrease in the expression transforming growth factor b (TGF-b) receptors.(52) Exposure to inhibitors of collagen synthesis, or a neutralizing antibody to a2b1, abolished these changes in ALP activity and TGF-b receptor expression.(52) This is consistent with Coffey’s maxim that “what a cell touches has a major role in determining what a cell does.”(45) Similarly, neutralizing the binding activity of the integrin avb3, critical to the osteoclast-matrix association, inhibited bone resorption in vitro and prevented osteoporosis in oophorectomized rats.(53) The osteoblast actin cy-

158 toskeleton is involved in mediating the response to fluid flow.(54) Mechanotransduction in bone may occur via loading-dependent flow of interstitial fluid through the haversian-canalicular network.(54) Osteoblasts, as well as osteocytes, in culture responded to pulsating fluid flow by release of enhanced amounts of prostaglandin E2 (PGE2).(54) Treatment of these cell preparations with cytochalasin B, an actin poison, abrogated the release of PGE2 in response to fluid flow.(54)

THE SUBSTRUCTURE OF THE NUCLEUS IS THE NUCLEAR MATRIX, A UNIQUE BIOCHEMICAL FRACTION THAT MEDIATES A CELL TYPE-SPECIFIC ORGANIZATION OF THE GENOME The mechanisms that underlie cytoskeletal-nuclear communication are not known, but the nuclear matrix is an attractive candidate for transmitting mechanical information between the cytosol and nucleus. This nucleoskeleton is operationally defined as the proteinaceous substructure that resists nuclease digestion and high-salt extraction.(55) Transmission electronmicrographs of the nonchromatin structure of the nucleus and analysis of this biochemical fraction indicate that the nuclear matrix includes the lamin– nucleopore complex, a dense fibrillogranular lattice of ribonucleoproteins and 9 –13 nm heteronuclear RNA (hnRNA) filaments, and the chromosome scaffold.(56 –59) This unique biochemical fraction of ribonucleoproteins and lamins is typically represented as a filamentous nuclear scaffolding or a network of channels connected with the nuclear pores.(45,60) However, the nuclear matrix appears to be more than an extension of the tissue matrix into the nuclear space. Like the cytoskeleton, there are nuclear matrix proteins common to all cells.(61– 64) These include proteins like NuMA, a component of the nuclear core filaments of the interchromatin net in interphase cells but an organizer of the microtubules of the mitotic spindle during mitogenesis.(65) The lamins, the outer shell of the nuclear matrix, form a meshwork of filaments that lines the inside surface of the inner nuclear membrane.(66) These proteins are involved in the assembly of the nuclear envelope and higher order chromosomal structures.(66,67) The germ cell–specific lamin B3 appears to mediate the reorganization of nuclear and chromosomal structures during meiotic divisions, and when the protein was expressed in somatic cells in culture, their nuclear morphology was transformed from spherical to hook-shaped.(68) The nuclear matrins are common proteins that are components of the internal nuclear matrix.(69) Also, like the cytoskeleton, the nuclear matrix is comprised of tissue- and phenotypespecific proteins.(57) Recently, numerous DNA-binding proteins unique to this biochemical fraction have been identified, and the nuclear matrix accommodates the transient localization of transcription factors from the chromatin.(39,70,71) Evidence from biochemical, molecular, and confocal microscopy studies have demonstrated roles for nuclear matrix proteins in DNA replication,(72) the processing and transport of RNA transcripts,(73) and transcrip-

BIDWELL ET AL. tional regulation.(74,75) The protein composition and organization of the nuclear matrix reflects and appears to mediate changes in cellular growth, differentiation, and transformation.(39,76 –78) Tumor-specific, nuclear matrix proteins have been characterized in prostate, breast, and bladder tissues.(79 – 81) The diagnostic and prognostic value of these proteins is under investigation and may be the molecular correlate to the pathologist’s observation that cancer is often heralded by changes in nuclear morphology.(82) The nucleoskeleton or nuclear matrix mediates the threedimensional organization of DNA.(39,72,83,84) Although every cell has a complete copy of the genome, the DNA is organized into topologically constrained loop domains which provide the structural basis for tissue- and phenotypic-specific gene expression (Fig. 2).(39,85–91) The eukaryotic nucleus contains over 2 m of DNA, requiring many levels of organization for the ordered packaging of the genome (Fig. 2). The sense and antisense strands of the B-form of the DNA molecule, which comprises the majority of the genome, are organized into the 2 nm right-handed double helix, consisting of two antiparallel strands of purines and pyrimidines. The base pairs (bp) are spaced 0.34 nm apart, and the helix makes a complete turn every 10 bp or 3.4 nm which results in 36° of helical twist.(39) A major and minor groove are formed by the B-form double helix, and these two grooves provide distinct surfaces with which proteins can interact. The double helix wraps around a core of histone proteins, the nucleosomes, to form the secondorder structure. In this “beads on a string” conformation, 200 bp of DNA is wound around each histone octamer core.(39) In the radial coil model of the metaphase chromosome, six nucleosomes coil in a solenoidal conformation to form the 30 nm filament.(39,85–91) This 30 nm filament is further organized into the loop domains, 60 –100 kbp in length, and are attached at their base to the chromosome scaffold(39) (Fig. 2). In the radial coil model, five loops (;300 kbp) are arranged into a radial array subunit. These radial arrays are then turned sideways and coiled to form 240 nm coils that stack to form the chromosome(39) (Fig. 2). It is the proteins of the nuclear matrix that mediate the formation of the topological and functionally distinct DNA loop domains.(39,59,72,83) These loops are fastened to the chromosome scaffold through matrix attachment regions (MARs), also known as scaffold attachment regions (SARs).(39,59,92) Although a variety of MARs have been described,(92) the archetypal MAR/SAR is 200 – 800 bp in length, often A/T-rich, and exhibits the characteristics of a chromatin boundary element. These gene boundaries act to insulate the transcriptional activity of a particular gene from positional influences or from the action of an enhancer from a neighboring gene.(93) Additionally, MARs and the proteins that associate with them participate in the activation of transcription.(74) The architecture of transcription includes the further organization of the nuclear matrix into an interchromatin net that orders splicing factors (snRNPs), hnRNP particles, and thin fibrils into interchromatin granule clusters (IGCs) connected by perichromatin fibrils.(94 –99) For the transcription of some genes, the proteins of the ICGs migrate to the


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FIG. 2. The organization of nuclear DNA. The radial coil model. A transcriptionally active loop is shown extended along the nuclear matrix. The loops are attached to the chromosome scaffold through matrix attachment regions (MARs). Transient interactions with the nuclear matrix may be mediated by nuclear matrix elements (NMEs).

gene’s location on the chromosome loop, where transcription and RNA-processing take place.(99) Conversely, for other genes, it is the DNA that moves closer to the ICG for transcription.(97) Transcription is also regulated by the organization of the chromatin, and the nuclear matrix integrates with the chromatin via proteins like CHD1, a novel DNA-binding protein that associates with MARs, localizes to both chromatin and nuclear matrix subfractions, and plays a significant role in the determination of chromatin architecture.(100) The nonrandom topography of transcription reflects the higher order structure of the genome. Interphase chromosomes occupy broad but discrete nuclear territories,(101,102) and active genes are typically localized to the internal nucleus rather than the nuclear envelope.(103) Finally, the small fraction of protein-coding genomic DNA is localized in the G-light bands of the chromosome.(94)

The osteoblast nuclear matrix The protein composition and organization of the osteoblast nuclear matrix reflects the bone cell phenotype.(76 –78) The nuclear matrix protein composition of osteoblasts derived from fetal rat calvaria was specific to successive stages of differentiation in vitro.(76) The two-dimensional sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDSPAGE) profiles of nuclear matrix proteins from the calvarial cultures revealed proteins specific to the proliferative, matrix synthesis, and matrix mineralization stages of devel-

opment.(76) Additionally, nuclear matrix proteins common to each of these stages of osteoblast development were observed. This would suggest that the bone cell progression from an osteoprogenitor to a mature osteoblast involves a reorganization of nuclear architecture.(76) Major changes in the expression and organization of the major structural nuclear matrix proteins accompany osteoblast development.(104,105) Confocal laser scanning microscopy revealed that NuMA exhibited a diffuse distribution throughout the central planes of the nucleus, excluding the nucleolus, in interphase osteoblasts and osteosarcoma cells,(104) but, as observed in other cell types,(65,84,106,107) localized to the mitotic spindle apparatus during metaphase and anaphase of the dividing bone cell.(104) Similarly, osteoblast expression of the nuclear matrix proteins topoisomerase II (topo II)-a and -b were determined to be specific to the stage of development in bone.(105) These proteins are major structural components of the mitotic chromosome scaffold and interphase nuclear matrix of all cells.(108 –110) The isoforms topo II-a and -b are products of two different genes(110,111–113); topo II-a expression is upregulated during mitogenesis, and topo II-b is typically expressed when cells have plateaued in growth.(110) Immunohistochemical analysis of histologic sections of lumbar vertebrae from 4- to 5-week-old male Sprague-Dawley rats indicated that topo II-a was expressed in cells of the bone marrow cavity, where the osteoprogenitor cells reside, and was conspicuously absent in mature osteoblasts and osteo-

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FIG. 3. Architectural transcription factors. Architectural transcription factors modulate promoter geometry. In the compass model, the architectural transcription factor bends the DNA and brings distal trans-acting proteins together, without interacting with the other regulatory proteins.(22) In the scaffold model, the DNA-bending proteins make contact with the other trans-acting proteins in the complex.(22) Architectural transcription factors can function by different mechanisms in different promoter contexts. cytes.(105) Topo II-b, however, was expressed in cells of the bone marrow cavity as well as mature osteoblasts and osteocytes.(105) The alteration of osteoblast DNA topology may precede and mediate the sweeping changes in gene expression that occur during the transition from an osteoprogenitor cell to a mature osteoblast. Consistent with a phenotype-specific osteoblast nuclear architecture, nuclear matrix and intermediate filament proteins specific to rat and human osteosarcomas have been characterized.(77,78) In the human studies, one-dimensional SDS-PAGE profiles of the major tissue matrix proteins revealed that the nuclear matrix and intermediate filament protein composition of normal osteoblasts did not vary with biopsy site, age, or gender of patients.(78) The loss of expression of an ;250 kDa intermediate filament protein designated as OB250 was observed in primary human osteosarcoma tumors and human osteosarcoma cell lines. A potential tumor-specific nuclear matrix protein (OT200) was also observed in the osteosarcoma tissue.(78) A redistribution of transcription factors between the osteoblast nuclear matrix and the chromatin, contributes to the change in the nucleoskeleton protein composition.(70) ROS 17/2.8 cells were observed to exhibit a cell typespecific distribution between the chromatin and nuclear matrix subfractions of the trans-acting proteins (or related DNA-binding activities) of SP-1, ATF, CCAAT, OCT-1, and AP-1.(70) The functional significance of the nuclear partitioning of trans-acting proteins has not been established but may mediate a localized concentration of these low-abundant proteins, thus favoring activation of transcription.(70,114)

NUCLEAR MATRIX, DNA-BINDING PROTEINS MAY TRANSDUCE CHANGES IN CELL SHAPE INTO CHANGES IN GENE ACTIVITY BY ALTERING PROMOTER GEOMETRY The cytoskeletal-nuclear communication pathway may involve a tissue matrix–induced alteration in genomic organization that ultimately leads to a change in promoter geometry. The molecular consequence of changing cell shape may be the twisting or bending of the promoters

coupled to the tissue matrix. This mechanical alteration of gene structure could contribute to a change in activity. DNA-binding, nuclear matrix proteins are in a unique position to couple cell and promoter structure. There are numerous DNA-binding, nuclear matrix proteins that associate with the MARs and appear to modulate gene expression through altering some aspect of DNA structure, putatively supercoiling.(34 –36,74) The AT-rich character of these sites confers an anomalous DNA structure that certain kinds of proteins recognize instead of a particular nucleotide sequence.(34,115) For example, ATC sequences are stretches of DNA in which one strand is A’s, T’s, and C’s, excluding G’s. These sequences of more than 20 bp are usually found in clusters within MARs and impart a strong unwinding ability to these sites.(34,35) Proteins such as SATB1 (special AT-rich sequence-binding protein),(34) Bright (B cell regulator of IgH transcription),(36,74) and nucleolin recognize the minor groove of these ATC/MAR sites and may mediate the relief of negative superhelical strain by stabilizing the base unpairing of these regions.(35) Bright, a B cell–specific protein, binds MAR sequences flanking the core of the intronic enhancer Em of the immunoglobulin heavy-chain (IgH) locus and is required for the activation of Em-driven IgH transcription.(36,74) The topo II enzymes are the most well characterized of the nuclear matrix, MAR-binding proteins.(108 –110) Topoisomerases alter DNA structure or topology by a concerted cleavage/religation reaction, which relaxes local supercoiling incurred during DNA replication and transcription.(108 –110) Changes in supercoiling influence the accessibility of DNA by transcription factors,(116 –118) and topo II cleavage sites are distributed throughout the genome, frequently in close proximity to gene enhancer regions.(119 –121) Topo II enzymes also interact directly with RNA polymerase II and specific transcription factors.(122,123) A recent hypothesis proposes the presence of multiple nuclear pools of topo II enzymes that regulate gene expression at the levels of the single and multiple genes.(124) Topo II may govern the expression of single-target genes by binding directly to their promoter regions, and, at the second level, MAR-bound topo II may exert a general effect on the transcriptional expression of clusters of genes by controlling supercoiling of chromosome loops.(124)

OSTEOBLAST NUCLEAR MATRIX Bending the DNA by architectural transcription factors is another mechanism by which gene activity is mechanically regulated.(21–33) The bending or looping of a promoter contributes to governing gene activity by altering the interactions between proximal and/or distal trans-acting proteins and the basic transcription machinery.(21–33) These architectural proteins often lack distinct activation domains, do not necessarily initiate transcription themselves, or interact with the other regulatory proteins.(21–33) As with MARbinding proteins, architectural transcription factors often recognize the minor groove of AT-rich regions of DNA which confers a local structural anomaly to the B-DNA helix.(115,125–127) High mobility group (HMG) proteins are an abundant, heterogeneous family of architectural transcription factors that comprise the nonhistone components of the chromatin.(22) LEF-1 and HMG-I(Y) are both HMG proteins that bind to the minor groove of DNA, induce DNA bending, and are required for enhancer activity but do not activate transcription on their own.(24,128 –135) LEF-1 binds to the T cell receptor a-gene enhancer and induces a sharp bend in the DNA, which brings together two nonadjacent sites, one binding to Ets-1, a lymphocyte-specific trans-acting protein, and the other to an ATF/CREB protein.(129,133–135) The bending protein does not interact with the other regulatory proteins in this “compass” structure(22) (Fig. 3). HMG-I(Y) binds to AT-rich sequences located in the center of a binding site for the dimeric NF-kB protein and adjacent to that for ATF-2 in the promoter of the human interferon b gene.(24,130 –132) Through DNA bending and protein–protein contacts, HMG-I(Y) mediates the formation of a higher order nucleoprotein complex between itself and these other trans-acting proteins, termed an enhanceosome.(24,130 –132) In this “scaffold” model,(22) the DNA-bending proteins make contact with the other proteins in the complex (Fig. 3).

Osteoblast, nuclear matrix, DNA-binding proteins Confocal laser scanning microscopy revealed that topo II enzymes exhibited varied and distinct nuclear distributions in bone cells,(105) consistent with this hypothesis of multifunctional nuclear pools.(124) Topo II-a exhibited a punctate distribution throughout the central portion of the nucleus of cycling ROS 17/2.8 rat osteosarcoma cells and rat primary spongiosa osteoblasts,(105) whereas the distribution of topo II-b was more diffuse, including a localization near the nuclear envelope in both cycling and noncycling cells.(105) Coupled with the differential expression of topo II-a and -b in the developing osteoblast, these nuclear matrix proteins may mediate distinct phenotype-specific MAR organizations, thus potentially regulating clusters of genes through supercoiling. Osteoblast, nuclear matrix, DNA-binding proteins, with a capacity for bending DNA, recognize elements on the osteocalcin and type I collagen promoters.(38,75,136 –138) This establishes a physical link between the regulatory regions of osteoblast ECM genes and cell structure. Osf2/Cbfa1, a transcriptional activator of osteoblast differentiation,(75,137,139 –141) was originally identified as NMP2, an osteoblast-specific, nuclear matrix protein.(38,142) Early

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FIG. 4. Nuclear matrix protein binding sites on the rat osteocalcin and type I collagen promoters. Overlapping recognition sites for NMP1/YY1 and NMP2/Cbfa1 are located at 2609/2587 nt and at 2457/2430 nt. This latter site contains an AP1/VDRE that mediates NMP1/YY1 repression of vitamin D induction of osteocalcin. The type I collagen cis-regulatory promoter element that mediates osteoblast expression in vivo is located within 21683/21670 nt, but the region between 23600 and 22300 nt is critical for expression in cultured bone cells. NMP3 is between 22149 and 22106 nt and is in proximity to a putative VDRE (22221 nt) and an AP-1 site (22250 nt). NMP4 is located in two AT-rich regions between 23469/23450 nt and 21574/21555 nt. Abbreviations: OC-box, osteocalcin box; GRE, glucocorticoid response element. studies indicated that this DNA-binding protein recognized sites along the rat and mouse osteocalcin promoters (Fig. 4); however, more recent work suggests that this transcription factor may regulate numerous genes including osteopontin and type I collagen (COL1A1).(75) Mice with a homozygous mutation to Cbfa1 died just after birth and lacked mature osteoblasts and a calcified skeleton.(139 –141) NMP2/Osf2/Cbfa1 belongs to the AML/CBF/PEBP2/Runt family of transcription factors which have the capacity to bend DNA.(38) Likewise, YY1, a ubiquitous DNA-binding/ bending protein,(30) was identified as NMP1 in bone cells and was observed to localize in both the chromatin and nuclear matrix fractions of osteoblasts and associated with sequence specificity to the osteocalcin promoter.(37,142) The molecular mechanisms by which NMP2/Osf2/Cbfa1 and NMP1/YY1 mediate osteocalcin transcription have yet to be elucidated, but because these proteins have the capacity to bend DNA, they may act to modulate osteocalcin promoter topology. It has also been proposed that NMP2/Osf2/ Cbfa1 and NMP1/YY1 may also serve to transiently tether the osteocalcin promoter to the nuclear matrix, thereby mediating the local concentration of trans-acting proteins.(114) Two distinct, osteoblast nuclear matrix, DNA-binding proteins, NMP3 and NMP4, associate with sequence-spec-

162 ificity to the rat type I collagen promoter(138) (Fig. 4). Interestingly, the type I collagen cis-regulatory promoter element that mediates osteoblast expression in vivo is located within 21719 and 21670 nucleotides (nt), but the region between 23600 and 22300 nt is critical for expression in cultured bone cells.(143,144) This peculiar dichotomy between transcriptional regulatory regions in vivo and in vitro has been attributed to differences in osteoblast structure within bone and on plastic.(145) NMP3 and NMP4 bind within or in close proximity to these regulatory regions.(138,146) NMP4 has many of the properties of an architectural transcription factor.(146) This nuclear matrix protein, along with an HMG-like, soluble nuclear protein, NP, was determined to bind within the minor groove of two poly(dT) sites between 23469/23450 nt and 21574/21555 nt of the COL1A1 promoter(138,146) (Fig. 4). Circular permutation analysis indicated that protein binding to these sites induced a bend in the DNA.(146) These poly(dT) sites are in proximity to both the in vivo and in vitro regulatory regions of the COL1A1 promoter and consist of a succession of nine uninterrupted thymidines, which are critical to mediating the nuclear matrix protein binding.(146) This stretch of thymidines (and adenines on the antisense strand) form a very narrow DNA minor groove which is attractive to architectural transcription factors.(115) NMP3 binding activity was observed between 22149 and 22106 nt. This region contains a CarG box, an element that has been implicated in DNA bending,(147) and is in proximity to other potential regulatory elements including a putative vitamin D response element (VDRE) (22221 nt) and an AP-1 site (22250 nt).(148) Both the NMP3 and NMP4 proteins exhibited a tissue-restricted distribution and were observed to be selectively expressed in osteoblasts or osteoblast-like cells.(138) NMP3 and NMP4 may link the shift in cis-regulatory elements that regulate the basal transcription of type I collagen between osteoblasts in situ and in culture and may couple osteoblast shape and promoter geometry.

THE NUCLEAR MATRIX IS A TARGET FOR HORMONE ACTION Numerous steroid hormone/receptor complexes, including those that play a significant role in bone metabolism (e.g., 1,25-dihydroxyvitamin D3, and estrogen) localize to the nuclear matrix in a variety of endocrine tissues.(39,45,71,149 –152) Cooperative interactions between nuclear matrix and chromatin proteins may mediate tissue specificity of ligand-induced response by governing the accessibility of target cis elements.(39,45,71) Mammary gland differentiation and involution are mediated by a cooperative signaling pathway between the tissue matrix and the peptide hormone prolactin.(7) This “solid-state” hormonal signaling pathway involves the restructuring of the cytoskeleton and nuclear matrix that allows the prolactin signal to activate transcription of the b-casein gene.(7,8)

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Parathyroid hormone, vitamin D, and the osteoblast tissue matrix Both 1,25-dihydroxyvitamin D3 (vitamin D) and parathyroid hormone (PTH) induce dramatic changes in osteoblast shape.(153–158) On the tissue level, these changes in osteoblast morphology may allow access of osteoclasts to the bone surface.(154) At the molecular level, the alteration of tissue matrix protein organization, including the nuclear matrix, may modulate promoter geometry and the accessibility to the regulatory elements of target genes.(71,159) The PTH-induced mobilization of the cyclic adenosine monophosphate (cAMP)-PKA pathway causes a rapid change in osteoblast shape, from a cuboidal to a stellate morphology.(153–158) This alteration in osteoblast shape includes major changes in the expression of structural proteins,(155,160 –163) a retraction of the cell’s peripheral lamellipodia, a massive reorganization of microfilaments, but not microtubules(160) and a decrease in the number of stress fibers.(155) In osteoblasts derived from fetal rat calvariae, PTH induced a rapid (2–5 minutes) dose-dependent (IC50 5 1 nM) depolymerization of actomyosin to about 50% of pretreatment levels attended by an equally rapid (2–3 minutes) dephosphorylation of myosin light chain.(155) These changes in cytoskeletal organization were transient, the levels of polymerized actin and phosphorylated myosin light chain returned to pretreatment levels within 30 minutes and 5 minutes, respectively.(155) Stable alterations in both the human and rat osteoblast cytoskeletons were observed with chronic exposure to PTH.(161–163) PTH selectively alters the bone cell expression of the nuclear matrix proteins topo II-a and NuMA.(104,163) Chronic exposure to hormone (10 nM) increased topo II-a and NuMA protein levels by a factor of two in the rat osteosarcoma cell cultures of ROS 17/2.8.(163) Topo II-b expression was not altered under the same experimental conditions.(163) Immunofluorescent and confocal laser scanning microscopy indicated that PTH increased the number of ROS 17/2.8 cells immunopositive for NuMA as opposed to an increase in NuMA protein/nucleus.(104) It was proposed that this change in NuMA expression, and thus nuclear organization, was coupled to the observed concomitant hormone-induced alterations in the bone cell cytoskeleton.(104) PTH also targets nuclear matrix proteins specific to osteoblast.(146,163) Acute and chronic exposure to PTH attenuated the expression of a 190 kDa nuclear matrix protein in both rat primary spongiosa osteoblasts and ROS 17/2.8 cells within 1 h.(163) This protein exhibited a restricted-tissue distribution and was not observed in opossum kidney (OK) cells, H4 hepatoma cells, or NIH3T3 mouse fibroblasts. Pulse-chase experiments indicated that the 190 kDa protein had a half-life of 1 h in the osteosarcoma cells.(163) These data are consistent with the hypothesis that the osteoblast nuclear matrix is a dynamic structure and is comprised, in part, of labile proteins that respond rapidly to hormonal signals. PTH modulated the DNA-binding activity of NMP4 along the type I collagen promoter.(146) Chronic exposure to hormone (10 nM, 72 h) up-regulated NMP4, but not

OSTEOBLAST NUCLEAR MATRIX NMP3, binding activity in ROS 17/2.8 cells and attenuated COL1A1 mRNA expression.(146) The nuclear matrix protein NMP1/YY1 repressed vitamin D–induced trans-activation of the osteocalcin gene in ROS 17/2.8 cells.(164) Osteosarcoma cells were cotransfected with an osteocalcin promoter-reporter construct (OC-CAT), containing VDRE, in combination with an NMP1/YY1 expression plasmid. NMP1/YY1 repressed vitamin D–induced activity of the OC-CAT construct but had no effect on the basal activity in the absence of vitamin D.(164) NMP1/YY1 repression of the OC promoter activity and vitamin D inducibility were lost using constructs lacking the VDRE. NMP1/YY1 recognition sequences were identified within the VDRE of the osteocalcin gene (Fig. 4). NMP1/YY1 and the vitamin D receptor/retinoid X receptor heterodimers competed for binding at the osteocalcin VDRE and competed for binding to TFIIB, an essential component of the transcriptional initiation complex.(164) How NMP1/YY1 interacted with TFIIB over a distance of 500 nt was not addressed in this study, but some form of DNA bending or looping would likely be required. Therefore, NMP1/YY1-induced changes in osteocalcin promoter geometry may contribute to the mechanisms underlying repression of vitamin D inducibility.

THE OSTEOBLAST NUCLEAR MATRIX PLAYS A REGULATORY ROLE IN GENE EXPRESSION AS CRITICAL AS THE CELL MEMBRANE The osteoblast nuclear matrix is a regulatory conduit through which changes in cell shape are translated into changes in gene activity, and reciprocally, the expression of the genetic program is rendered into three-dimensions. By altering DNA organization and structure, this complex nucleoskeleton is uniquely placed to convert bone cell morphology into promoter geometry (i.e., gene activity) as well as to orchestrate the genetic program of the developing osteoblast by coordinating global changes in DNA organization that mediate the transitions between stages in bone cell development. The nuclear matrix provides a functional paradigm that is particularly useful for an understanding of the cell mechanics of osteoblast response to loading and PTH. The structural continuum from the ECM to the geometry of the promoter may directly link osteoblast deformation, induced by loading or PTH, with a realignment of those genes coupled to the tissue matrix and literally “tug” genes into or out of the genetic program. The study of the osteoblast nuclear matrix brings a topography to the nuclear events that regulate bone cell biology, and a molecular basis to osteoblast structure.

ACKNOWLEDGMENTS This work was supported by National Institutes of Health (NIH) grant R55 DK48310 (J.P.B.) and NIH grant RO1 DE7272 (J.M.H.).

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Address reprint requests to: Joseph P. Bidwell Indiana University School of Dentistry 1121 West Michigan Street Indianapolis, IN 46202 U.S.A. Received in original form January 24, 1997; in revised form August 8, 1997; accepted October 8, 1997.