Nmp4/CIZ regulation of matrix metalloproteinase 13 (MMP-13) response to parathyroid hormone in osteoblasts
Rita Shah,1
Marta Alvarez,1
Daniel R. Jones,2
Kitti Torrungruang,3
Andrew J. Watt,4
Nagarajan Selvamurugan,5
Nicola C. Partridge,5
Cheryl O. Quinn,6
Fred M. Pavalko,7
Simon J. Rhodes,8 and
Joseph P. Bidwell1
Departments of 1Anatomy and Cell Biology and 7Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis 46202; 2Division of Natural Sciences and Mathematics, Indiana Wesleyan University, Marion 46953; 8Department of Biology, Indiana University-Purdue University Indianapolis, Indiana 46202; 3Faculty of Dentistry, Department of Periodontology, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand; 4University of Michigan Medical School, Ann Arbor, Michigan 48109; 5Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5627; and 6Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110
Submitted 14 November 2003
; accepted in final form 9 March 2004
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ABSTRACT
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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 PGE2. 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.
collagen; collagenase; bone; endocrine
PARATHYROID HORMONE (PTH) induction of matrix metalloproteinase-13 (MMP-13, also called collagenase-3) expression in osteoblasts is a key step in the process of normal bone turnover (33). Bone remodeling, endochondral bone formation, and bone repair all involve osteoblast expression of this enzyme (31, 38, 39). MMP-13 has a wide range of substrates, including types I, II, and III collagen, gelatin, aggrecan, perlecan, fibrillin, and the large isoform of tenascin C and thus also plays a significant role in tumor invasion and metastasis (20, 21). Both transcriptional and posttranscriptional mechanisms stringently control the expression and activity of this potent MMP (20).
PTH responsiveness of the MMP-13 gene is mediated by the region spanning nucleotides (nt) 38 through 148 (Fig. 1 and Refs. 10, 14, 25, 2729). This region contains two cis-elements for the transcription factor Runx2, a master regulator of osteoblast differentiation (17), and cis-elements for activator protein (AP)-1, PEA-3/Ets-1, and p53, all conserved among the rat, mouse, and human genes (Fig. 1). Recent studies indicate that combinatorial interactions of these cis-regulatory elements govern osteoblast expression of MMP-13 (10, 14, 24, 25, 29). How these multiple associations are integrated and the identities of all of the trans-acting factors interacting with these elements have not been established.

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Fig. 1. Schematic representation of the rat matrix metalloproteinase-13 gene (MMP-13) 5'-parathyroid hormone (PTH)-responsive region from +1 through 132 nucleotides (nt). A homopolymeric(dA:dT) element is located at 119/110 nt, between the distal Runx2 (137/126 nt) and activator protein (AP)-1 (48/42 nt) consensus sites that are critical for hormone response and basal transcription (10, 14 ,25, 2729). The elements shown in gray are conserved across the rat, mouse, and human MMP-13 5'-regulatory regions.
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A homopolymeric(dA:dT) element within the MMP-13 PTH response region conforms to the nuclear matrix protein-4/cas interacting zinc finger protein (Nmp4/CIZ) consensus sequence (Fig. 1 and Refs. 23 and 35). Nmp4/CIZ are novel nucleocytoplasmic shuttling transcription factors, independently characterized in the nuclear matrix (NM) and soluble nuclear (NE) fractions of osteoblasts (3, 35) and in the cytoplasm of fibroblasts associating with the docking/adaptor protein p130cas (23).
Nmp4/CIZ is expressed in many cell types but has a role in regulating the development of osseous tissue. Overexpression of Nmp4/CIZ in MC3T3-E1 osteoblast-like cells represses BMP2-enhanced expression of osteoblast phenotypic genes and attenuates the formation of mineralized nodules in cell cultures (30). These proteins are highly expressed in rodent embryonic bone (35), and an Nmp4/CIZ element of the type I collagen
1(I) polypeptide chain (COL1A1) gene may be associated with bone mineral density in humans (12). Finally, consistent with a role in extracellular matrix turnover, Nmp4/CIZ supports transcription of the MMP-1, -3, and -7 genes in fibroblasts (23, 37).
In the present study, we undertook to determine whether Nmp4/CIZ contributes to osteoblast expression of MMP-13. We find that Nmp4/CIZ binds to the homopolymeric(dA:dT) element within the MMP-13 PTH response region and supports basal transcription. Furthermore, Nmp4/CIZ and Runx2 act as components of a synergy control element to regulate the amplitude of the MMP-13 response to PTH. These data, together with previous studies, suggest that Nmp4/CIZ is an integral part of an enhanceosome governing MMP-13 expression.
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MATERIALS AND METHODS
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Cell culture.
UMR 106-01 rat osteosarcoma cells and MC3T3-E1 murine osteoblast-like cells (provided by Dr. Laurie McCauley, University of Michigan, Ann Arbor, MI) were grown in either MEM or
-MEM (GIBCO-BRL, Grand Island, NY), respectively. Media were supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 25 µg/ml amphotericin, 2 mM L-glutamine (GIBCO-BRL), and 10% FBS (Sigma, St. Louis, MO). ROS 17/2.8 cells, kindly donated by Drs. Gideon and Sevgi Rodan (Merck Research Laboratories, West Point, PA), were grown in Ham's F-12 medium supplemented with 2.36 g/l NaHCO3, 0.118 g/l CaCl2·2H2O, and 6.106 g/l HEPES, as well as the supplements described above. NIH-3T3 murine fibroblasts were grown in DMEM (high glucose; GIBCO-BRL) supplemented with 10% bovine calf serum (Sigma) and penicillin, streptomycin, amphotericin, and L-glutamine, as described above. All cells were maintained in humidified 95% air-5% CO2 at 37°C.
MMP-13 promoter-reporter constructs.
The rat MMP-13-CAT promoter-reporter construct containing the first 1329 nt of the 5'-regulatory region (here referred to as WTCL3-CAT) has been described in earlier studies (26, 27). To determine the 5'-regulatory sequence beyond the first 456 nt already reported (27), the insert of the WTCL3-CAT vector was sequenced by automated fluorescent DNA sequencing. Initially, the T7 promoter primer, which anneals 5' to the insert on the pBluescript SK vector backbone, was used. Additional sequencing primers were designed to complete sequencing to the 3' end of the insert. The sequence from 1329 to 456 nt was submitted to GenBank with accession number AY135636.
The rat MMP-13-CAT promoter-reporter construct containing a 3 A-to-C mutation of the Nmp4/CIZ site (119/110 nt; here referred to as MUTNCL3-CAT) was made using the Quick Change PCR Site-directed Mutagenesis Kit (Stratagene) and the following mutagenic primers: 5'-CTCAGGTTCTGCCACAAACCACACGTACGAAAACCCAAATACCACGTAAGCATG-3' (sense); 5'-CATGCTTACGTGGTATTTGGGTTTTCGTACGTGTGGTTTGTGGCAGAACCTGAG-3' (antisense).
The rat MMP-13-CAT promoter-reporter construct MUTRCL3-CAT contained the following mutations (underlined) in the Runx2 site (137/126, adapted from Ref. 14): GTACCAAGAACA. The MUTRNCL3-CAT promoter-reporter construct contained the described mutations in both the Runx2 and Nmp4/CIZ sites. The sequence fidelity of each construct was determined as described above.
Cell transfections.
UMR 106-01, ROS 17/2.8, MC3T3-E1, and NIH-3T3 cells were transfected with the MMP-13 promoter-reporter constructs using Lipofectamine Plus (Life Technologies, Gaithersburg, MD). Transfected cells were selected with Geneticin (Life Technologies), selected for 34 wk, and collected as pools (300600 colonies/construct). To determine the effect of overexpressing Nmp4/CIZ and other trans-acting proteins on MMP-13 transcription, the UMR 106-01 cells, stably transfected with WTCL3-CAT, were transiently transfected with either the empty pcDNA3 expression vector (Invitrogen, Carlsbad, CA) or this plasmid encoding the protein of interest.
PTH and PGE2 treatments.
UMR 106-01, MC3T3-E1, or ROS 17/2.8 cells, stably transfected with MMP-13 promoter-reporter constructs, were seeded in six-well culture plates and grown to confluence. Cells were treated with 10 nM rat PTH-(134) (Bachem, Torrance, CA) or the same volume of vehicle (10 mM acetic acid) for 672 h. For the PGE2 experiments, cells were incubated in serum-free medium for 24 h before challenge with either PGE2 (Sigma) or the same volume of vehicle (0.1% ethanol) for 624 h. Cells were then harvested and analyzed for CAT activity as described below.
CAT assays and statistical analysis.
Cells were harvested with the CAT Enzyme Assay System (Promega, Madison, WI) and analyzed for CAT activity according to the manufacturer's instructions. Chloramphenicol, D-threo-[dichloroacetyl-1,2-14C]CAT Assay Grade, was obtained from NEN/Perkin-Elmer (Boston, MA). Total protein concentrations were determined using the Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL). Raw data for the CAT assays were collected as disintegrations per minute per microgram of protein per hour. For statistical analysis and comparison between multiple experiments, these data were typically converted either to percentage of wild-type control, for comparison of basal transcriptional activities, or to degree of response over vehicle-treated cells, for comparison of hormone and prostaglandin degree of induction. Statistical analyses were performed by using one-way ANOVA incorporating a Student's t-test (JMP Statistical Software, version 4.0.4, SAS Institute, Cary, NC).
Protein extract and lysate preparations.
NM proteins were prepared using a sequential extraction protocol (35). A standard method (11) was employed for obtaining NE extracts.
Electrophoretic mobility shift assays.
Protein-DNA interactions were characterized using electrophoretic mobility shift assay (EMSA; see Refs. 2 and 35). Binding reactions were performed in a total volume of 20 µl and contained 100 mM KCl, 20% glycerol, 0.2 mM EDTA, 20 mM HEPES (pH 7.9), 0.01% Nonidet P-40, 0.5 mM dithiothreitol, 2 µg poly(dI:dC), 0.52 µg of NM protein, and 0.5 nM end-labeled rat MMP-13 or Klenow fill-in-labeled COL1A1 5'-regulatory regions as the probe. For experiments designed to identify the Nmp4/CIZ protein-DNA interactions, either 0.5 µl of preimmune serum (normal rabbit control IgG; Santa Cruz Biotechnology, Santa Cruz, CA) or 0.5 µl of polyclonal Nmp4/CIZ antibody (35) was incubated for 30 min on ice with 0.5 µg of protein. Subsequently, the poly(dI:dC) and end-labeled probe were added for a further 30-min incubation at room temperature. EMSA binding reactions were separated on 8% nondenaturing polyacrylamide gels (80:1 acrylamide-N,N'-methylbisacrylamide) in 1x Tris-glycine EDTA buffer (4°C). The gels were dried under vacuum at 80°C for 1 h. Kodak X-AR film was exposed to the gel with an intensifying screen overnight at 80°C.
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RESULTS
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Nmp4/CIZ binds with sequence specificity to the 5'-regulatory region (119/110 nt) of rat MMP-13. Nmp4/CIZ proteins bind to homopolymeric(dA:dT) elements (23, 35). Sequence analysis of the first 1329 nt of the rat MMP-13 5'-regulatory region revealed that the most extensive homopolymeric(dA:dT) tract is between 119 and 110 nt, with 10 consecutive adenine bases (accession no. AY135636). This site is within the PTH-responsive region of this gene (Fig. 1 and Refs. 10, 14, 27, 29). To determine whether Nmp4/CIZ proteins recognize this homopolymeric(dA:dT) sequence, we used EMSA to first ascertain whether sequence-specific DNA-binding activity could be recovered in the NE protein and/or NM protein fractions of UMR 106-01 osteoblast-like cells using a DNA probe spanning 137 to 107 nt (WTCL3137/107; Fig. 2). We observed distinct NE and NM sequence-specific protein-DNA binding profiles along this region of the MMP-13 gene (Fig. 2). Addition of an Nmp4/CIZ polyclonal antibody to the NE-DNA binding reactions resulted in a supershift band (Fig. 3A). A similar Nmp4/CIZ antibody supershift was observed in the NM fraction (data not shown). The presence of an antibody to Runx2 in these NE-DNA binding reactions also induced a supershift band (Fig. 3A), consistent with previous studies (10, 14). Similar observations were recorded in the EMSA profiles of the NM-DNA binding reactions (data not shown). Mutation of the MMP-13 homopolymeric(dA:dT) element (MUTNCL3137/107) abolished the Nmp4/CIZ antibody-induced supershift (Fig. 3B). Interestingly, the Runx2 antibody-induced supershift appeared more intense along this mutated probe (Fig. 3B). Similarly, mutation of the MMP-13 Runx2 element (MUTRCL3137/107) not only abrogated the antibody-induced supershift but enhanced the Nmp4/CIZ antibody-induced supershift (Fig. 3C). Finally, mutation of both sites along the MMP-13 gene (MUTRNCL3137/107) eliminated binding of both Nmp4/CIZ and Runx2, as demonstrated by a lack of supershift bands upon the addition of antibodies (Fig. 3D).

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Fig. 2. Sequence-specific soluble nuclear (NE) and nuclear matrix (NM) protein-DNA binding activity is observed within the PTH-responsive region of the MMP-13 gene containing the homopolymeric(dA:dT) element. Electrophoretic mobility shift assay (EMSA), using NE and NM extracts from UMR 106-01 cells, demonstrates a sequence-specific DNA binding activity between 137 and 107 nt (probe) of the MMP-13 gene (WTCL3137/107). These protein-DNA interactions are competed for by 100x cold specific probe (Sp Comp) but are unaffected by 100x nonspecific DNA (Ns Comp). Note the distinct NE- and NM-DNA binding profiles.
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Fig. 3. Nuclear matrix protein-4/cas interacting zinc finger protein (Nmp4/CIZ) binds to the homopolymeric(dA:dT) element within the PTH-responsive region of the MMP-13 gene. EMSA analysis with variations of the MMP-13 (CL3) gene 5'-regulatory region 137/107 nt as the probe. Data for the wild-type probe (WTCL3137/107; A), probe containing a mutated homopolymeric(dA:dT) element (MUTNCL3137/107; B), probe containing a mutated Runx2 element (MUTRCL3137/107; C), and probe containing both mutated homopolymeric(dA:dT) and Runx2 elements (MUTRNCL3137/107; D) are shown. Black circles indicate antibody-induced supershifts. Ab, antibody; IgG, preimmune serum for Nmp4/CIZ Ab; NE, soluble nuclear extract from UMR 106-01 cells.
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PTH regulates Nmp4/CIZ-DNA binding activity.
To determine whether PTH regulates Nmp4/CIZ binding within the MMP-13 hormone response region, we performed EMSA using this segment of the gene as a probe and nuclear extracts from cells treated with hormone or vehicle (Fig. 4). Consistent with a previous study using ROS 17/2.8 osteoblast-like cells (2), exposure of UMR 106-01 cells to 10 nM rPTH-(134) for 2472 h increased NM Nmp4/CIZ binding activity along the COL1A1 gene containing the homopolymeric(dA:dT) site (Fig. 4A). Some increase in binding was also observed in the NE fraction (Fig. 4A). This PTH-induced alteration in NE protein-DNA binding was not apparent along the MMP-13 region between 137 and 107 nt unless Runx2 binding was abrogated via the introduction of a mutation in the distal Runx2 binding site, adjacent to the Nmp4/CIZ element (Fig. 4B).

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Fig. 4. PTH alters Nmp4/CIZ-DNA binding. A: EMSA was used to characterize the binding activity of NM and NE extracts along the type I collagen 1(I) polypeptide chain (COL1A1) gene between 1594 and 1541 nt (WTCOL). Extracts were derived from UMR 106-01 cells treated with 10 nM rPTH-(134) or vehicle (VEH, 10 mM acetic acid) for 72 h. B: EMSA analysis with variations of the MMP-13 (CL3) gene 5'-regulatory region 137/107 nt as probes shows no PTH-induced increase in NE protein binding along the wild-type probe (WTCL3) or the probe containing a mutated homopolymeric(dA:dT) element (MUTNCL3). There is a hormone-induced increase in NE protein binding along the probe containing a mutated Runx2 element (MUTRCL3). This PTH-responsive band was supershifted upon addition of an Nmp4/CIZ antibody (C), confirming its identity.
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Nmp4/CIZ promotes basal MMP-13 transcription.
To determine whether Nmp4/CIZ binding contributes to basal activity of the MMP-13 promoter, we mutated the homopolymeric(dA:dT) site (119/110 nt) within the +34 to 1329 nt MMP-13 5'-regulatory region. Promoter-reporter constructs containing either the wild-type Nmp4/CIZ binding site (WTCL3-CAT) or the Nmp4/CIZ binding element mutation (MUTNCL3-CAT) were stably transfected into osteoblast-like cell lines and murine NIH-3T3 fibroblasts. Basal promoter-reporter activity was attenuated 4070% in the MUTNCL3-CAT constructs compared with WTCL3-CAT controls in UMR 106-01 and ROS 17/2.8 rat osteosarcoma cells, murine MC3T3-E1 osteoblast-like cells, and NIH-3T3 fibroblasts (Fig. 5A). Consistent with these results, overexpression of Nmp4/CIZ increased basal transcription of WTCL3-CAT in UMR 106-01 osteoblast-like cells approximately two- to threefold (Fig. 5B). Similarly, overexpression of Nmp4/CIZ in transiently transfected NIH-3T3 cells increased basal transcription approximately twofold (data not shown).

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Fig. 5. Nmp4/CIZ supports basal transcription of MMP-13. A: mutation of the MMP-13 Nmp4/CIZ consensus element attenuates basal activity of the promoter. Substituting 3 A nucleotides for C nucleotides between 119 and 110 nt reduced MMP-13 promoter-reporter activity (MUTNCL3-CAT) compared with wild-type controls (WTCL3-CAT) in a variety of stably transfected cell types. Statistical analysis indicates P < 0.0001 for all cell types except UMR 106-01, where P < 0.05; bars represent average of %WTCL3-CAT activity of 3 replicates ± SE. B: overexpression of Nmp4/CIZ (isoform 11H DNA; see Ref. 35) enhances relative CAT activity of WTCL3-CAT 1.5- to 3-fold in UMR 106-01 osteoblasts (P < 0.0001). Bars represent average of WTCL3-CAT activity (dpm/µg protein) of 3 replicates ± SE: open bars represent the activity of control cells transfected with the empty expression vector pcDNA3; filled bars represent the activity of cells transfected with the Nmp4/CIZ 11H.
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Nmp4/CIZ attenuates the PTH-induced increase in MMP-13 transcription.
To investigate whether Nmp4/CIZ plays a role in PTH-induced changes in MMP-13 transcription, osteoblast-like cells stably transfected with these promoter-reporter constructs were treated with the hormone or vehicle, and the CAT activity was measured. Treatment of UMR 106-01 cells with 10 nM rPTH-(134) for 6 h resulted in an approximately twofold induction in wtCL3-CAT activity (Fig. 6A), consistent with a previous study using transient transfections and a shorter promoter (29). By contrast, mutation of the Nmp4/CIZ element (MUTNCL3-CAT) did not attenuate the PTH response but instead resulted in a nearly fivefold induction in activity by hormone (Fig. 6A). Similar results were obtained with ROS 17/2.8 cells (data not shown). PGE2, another potent mediator of bone turnover that, like PTH, activates the osteoblast cAMP-protein kinase A response limb (7), had similar affects on WTCL3-CAT and MUTNCL3-CAT activity. PGE2 increased WTCL3-CAT transcription
2-fold and MUTNCL3-CAT activity by
4.6-fold (Fig. 6A).
Transcriptional synergy is the greater-than-additive activity that is a common form of interaction among activators recruited to regulatory regions comprised of multiple response elements (32). Our results suggest the hypothesis that Nmp4/CIZ mediates synergy control between the elements of the MMP-13 gene region involved in PTH hormone induction. Furthermore, Nmp4/CIZ and Runx2 are often organized in close proximity to each other along extracellular matrix and MMP gene regulatory regions (6). To determine whether Runx2 contributes to Nmp4/CIZ synergy control of MMP-13 hormone induction, we mutated both the Nmp4/CIZ and distal Runx2 consensus sites. For these experiments, we included an MMP-13 promoter-reporter construct that contained a Runx2 element mutation (MUTRCL3-CAT; see Ref. 14). We also tested an MMP-13 promoter-reporter construct that contained mutations in both the Nmp4/CIZ and Runx2 elements (MUTRNCL3-CAT). Hormone treatment [rPTH-(134), 10 nM, 24 h] of UMR 106-01 cells, stably transfected with these constructs, resulted in a 2-fold induction in wtCL3-CAT activity, an
4-fold induction in MUTNCL3-CAT activity, but only a 1.4-fold induction in MUTRCL3-CAT expression (Fig. 6B). The slightly attenuated PTH response of the MUTRCL3-CAT construct was not statistically significant but was consistent with earlier studies showing a similar effect on hormone responsiveness from mutating this element alone (29). Most striking, however, was the
11-fold induction in MUTRNCL3-CAT activity in response to PTH (Fig. 6B). Interestingly, introducing mutations in both the Runx2 and Nmp4/CIZ sites has no significant effect on the basal transcriptional activity of the MMP-13 promoter in UMR 106-01 cells (data not shown). Finally, overexpression of Nmp4/CIZ modestly attenuated the PTH-induced increase in WTCL3-CAT activity in UMR 106-01 cells. Cells transfected with 1 µg pcDNA3 exhibited a 2.4 ± 0.03-fold increase in WTCL3-CAT activity with hormone treatment (10 nM, 24 h; mean ± SE), whereas those cells transfected with an equal amount of the Nmp4/CIZ construct exhibited a 1.7 ± 0.17-fold increase in promoter-reporter activity (P = 0.03). The overexpression of Runx2 did not influence PTH induction nor did it augment Nmp4/CIZ-mediated attenuation of the hormone response (data not shown).
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DISCUSSION
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The present data demonstrate that Nmp4/CIZ binds within the PTH response region of the rat MMP-13 gene (10, 14, 25, 29), supports basal transcription, and contributes to synergy control of hormone induction. Mutation of this site abrogates Nmp4/CIZ binding to the PTH response region and decreases MMP-13 basal transcription in osteoblast-like cell lines and NIH-3T3 fibroblasts. Likewise, overexpression of Nmp4/CIZ enhances wild-type promoter basal activity. However, this same mutation of the homopolymeric(dA:dT) element enhances MMP-13 PTH and PGE2 responsiveness, and, consistent with these results, overexpression of Nmp4/CIZ modestly blunts hormone induction. Remarkably, mutation of the Nmp4/CIZ site and the adjacent Runx2 element dramatically augments the MMP-13 hormone response compared with mutation of the homopolymeric(dA:dT) element alone. However, overexpression of Runx2 did not influence PTH induction and, concordant with a previous study (29), neither did mutation of the distal Runx2 element alone.
Previous studies indicate that the MMP-13 PTH response region consists of multiple positive and negative regulatory elements that, through combinatorial interactions, mediate basal transcription and hormone induction (10, 14, 25, 29). For example, mutation of either the distal Runx2 element (132/126 nt; Fig. 1) or the AP-1 site (62/55 nt; Fig. 1) does not significantly influence promoter activity, but mutation of both elements abolishes basal and PTH-induced transcription (29). Additionally, Runx2 and c-Fos·c-Jun physically interact and cooperatively bind the AP-1 and distal Runx2 elements that are organized in a required helical arrangement within the MMP-13 PTH response region (10, 14). The proximal Runx2 element also contributes to MMP-13 PTH response, but whether it is involved in transcriptional synergy with the AP-1 site is not clear (14).
The dependence of MMP-13 PTH responsiveness on the stereospecific arrangement of multiple activator recognition sites within the hormone response region, cooperative binding between the trans-acting proteins within this region, and synergy between elements are the characteristics of an enhanceosome (8, 22). These higher-order nucleoprotein complexes are a means by which the cell, using a limited repertoire of activators, assembles combinations of ubiquitous and tissue-specific trans-activators to execute multiple regulatory decisions (8, 22). The aggregate transcriptional response, however, is not the simple arithmetic sum total of the independent effect of individual regulators but the integration of a complex set of operations that rely on combinatorial interactions between the proteins of the enhanceosome (16). This may account for our observation that mutation of both the Nmp4/CIZ and distal Runx2 sites has a more profound impact on hormone induction than the overexpression of both these proteins. Nmp4/CIZ is the only known Cys2His2 zinc finger protein that binds within the minor groove of poly(dA:dT) DNA, a property that may be critical to its capacity for altering DNA structure (2, 37). Introduction of nucleotide mutations that alter the AT-rich minor groove and flanking sequence may disrupt the underlying DNA architecture of the putative enhanceosome, effectively disabling synergy control, whereas simply increasing the local concentration of component proteins may have little impact on a mechanism that mediates fine control over hormone response amplitude.
Transcription factor modular organization likely underlies the capacity of the distal Runx2 element to mediate MMP-13 hormone induction, in association with the AP-1 site, while concomitantly effecting synergy control of this induction with Nmp4/CIZ. The typical trans-acting protein is comprised of distinct and separate transcriptional activation, repression, and DNA binding domains that can carry out multiple and sometimes opposing operations within the same enhanceosome (4, 36). For example, interferon regulatory factor-1, a component of the enhanceosome that activates the interferon (IFN)-
gene, has both activating and repression domains that may "contribute to balanced or tuned regulation of gene activation" (18). Nmp4/CIZ and Runx2 have both distinct transactivation and repression domains (34, 37) consistent with each protein's capacity for executing divergent functional roles in regulating MMP-13 transcription.
We are currently investigating the mechanisms underlying Nmp4/CIZ synergy control. This mode of transcriptional regulation, distinct from repression, is clearly complex. In some instances corepressors appear to vie with coactivators for activation domain association, thus disrupting synergy between DNA regulators without turning off the gene (9). The activation function-2 (AF-2)-binding protein, RIP140, strongly inhibits both Pit-1/thyroid receptor and Pit-1/estrogen receptor synergistic control of the growth hormone and prolactin promoters, respectively (9). It has been proposed that RIP140 and a hypothetical strong endogenous coactivator compete for binding to the AF-2 region of the nuclear receptor partner. In a second mechanism, the amino acid sequence, P-x(0,3)-I/V-K-x-E-x(0,3)-P, has been identified as a synergy control (SC) motif, distinct from activation domains in steroid receptors, the Sp1, Myb, and C/EBP families (16, 32). These domains are thought to recruit synergy control factors, and one such candidate is SUMO, a ubiquitin-like protein that may limit higher-order interactions among the transcription factors within the enhanceosome (19, 32). However, analysis of Nmp4/CIZ and Runx2 primary sequences has failed to yield strong candidates for SC motifs in these proteins. Interestingly, the DNA-binding sites of Nmp4/CIZ and Runx2 are often organized in close proximity to each other along extracellular matrix and MMP gene regulatory regions (6), thus perhaps comprising a general synergy control element of genes involved in bone turnover. The in vitro assembly of this putative PTH-responsive enhanceosome will be necessary to fully elucidate the multiprotein interactions involved in transcriptional control and to guide future experiments investigating these interactions in the context of the native chromatin (8).
There are alternative hypotheses to the synergy control model. For example, the poly(dA:dT) site within the MMP-13 PTH response region likely supports the association of the architectural transcription factor HMGI/Y (also called HMGA; see Ref. 15) and Nmp4/CIZ; thus these two proteins may contend for this binding site as the basis of a competitive regulation mechanism. HMGA binds to the minor groove of poly(dA:dT) DNA elements via an AT-hook motif (15). Interestingly, Nmp4/CIZ also has a putative AT-hook yet binds to the minor groove of this site via its zinc fingers (37). This competitive regulation hypothesis is consistent with the known role of HMGA in recruiting transcription factors to the nascent IFN-
enhanceosome (40) and with a role for HMGA as a repressor of transcription via competitive binding to AT-rich homeodomain enhancer elements (5).
The expression of MMP-13 during PTH-induced bone remodeling must be coordinated in both time and space and involves the stringent control of periodic activation and repression of transcription. The PTH enhanceosome localizes hormone control of this gene. The reciprocal interactions of multiple trans-acting proteins not only mediate hormone induction but also simultaneously restrain the magnitude of response, which is critical for regulating the potency and wide range of MMP-13 action, as well as confining rapid extracellular matrix remodeling.
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GRANTS
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This study was supported by National Institutes of Health Grants AR-45218 and DK-53769 (J. P. Bidwell), DK-47420 (N. C. Partridge), and HD-42024 (S. J. Rhodes) and National Science Foundation Grant 0131702 (S. J. Rhodes).
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FOOTNOTES
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Address for reprint requests and other correspondence: J. P. Bidwell, Dept. of Anatomy and Cell Biology, Indiana Univ. School of Medicine, Medical Science Bldg. 5035, 635 Barnhill Drive, Indianapolis, IN 46202 (E-mail: jbidwell{at}iupui.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES
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