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The nucleocytoplasmic shuttling transcription factor Nmp4/CIZ (nuclear matrix protein 4/cas interacting zinc finger protein) is a ubiquitously expressed protein that regulates both cytoplasmic and nuclear activities. In the nucleus, Nmp4/CIZ represses transcription of genes crucial to osteoblast differentiation and genes activated by various anabolic stimuli, including parathyroid hormone (PTH). We investigated the role of Nmp4/CIZ in the PTH-induced increase in bone by engineering mice with loss-of-function mutations in the Nmp4/CIZ gene, and treating 10-week-old female mice with anabolic doses of human PTH (1–34) at 30 μg/kg/day, 7 day/week, for 7 weeks or vehicle control. The untreated, baseline phenotype of the Nmp4-null mice between 8 and 16 weeks of age included a modest but significant increase in bone mineral density (BMD) and bone mineral content (BMC) compared to wild-type (WT) mice. Type I collagen mRNA expression was moderately elevated in the femurs of the Nmp4-null mice. The Nmp4 mutant alleles decreased body weight by 4% when expressed on a mixed background but the same alleles on a pure B6 background yielded a significant, 15% increase in body weight among the KO mice, compared to their WT controls. Hormone treatment equally enhanced BMD and BMC over vehicle-treated mice in both the WT and Nmp4-null groups but Nmp4-KO mice exhibited a significantly greater PTH-induced acquisition of femoral trabecular bone as compared to WT mice. These data support our hypothesis that Nmp4/CIZ is a transcriptional attenuator that suppresses osteoid synthesis and PTH-mediated acquisition of cancellous bone.
An accumulating body of clinical studies demonstrates that once-daily intermittent doses of human parathyroid hormone (1–34) [hPTH (1–34)] reduce the fracture risk in patients with severe osteoporosis (Dempster et al., 2001; Neer et al., 2001). Despite its proven efficacy in reducing fractures, and subsequent FDA approval as an osteoporosis therapy, the cellular and molecular mechanisms of action employed by intermittent PTH remain to be fully elucidated. Upon binding to its receptor (PTHR1), PTH activates a multitude of osteoblast molecular signaling networks, beginning with the cAMP/PKA and the cytosolic calcium/PKC response limbs. Subsequent downstream targets include the activation of the transcription factors CREB and Runx2, downregulation of the osteocyte-derived bone formation inhibitor sclerostin, and initiation of an IGF-1 autocrine/paracrine signaling loop, among others (Feister et al., 2000; Bikle et al., 2002; Swarthout et al., 2002; Hurley et al., 2006; Yakar et al., 2006; Zhang et al., 2006; Leupin et al., 2007; Li et al., 2007; Merciris et al., 2007). The collective end result of these and other PTH-induced networks is the enhanced differentiation of committed osteoblast precursors in the bone marrow, and prolongation of the lifespan of the mature osteoblast via suppressed apoptosis (reviewed in Jilka, 2007; Martin and Seeman, 2007).
While much of the investigation of anabolic PTH signal transduction and molecular regulation has been focused on the activation of osteogenic kinases, secretion of osteogenic ligands and ECMs, and expression of osteogenic genes, little is known about the inhibitory mechanisms on bone formation that are also activated by the anabolic signaling events. These mechanisms, though antagonistic to the bone-building effects of the signal, likely serve as an important negative feedback mechanism to prevent hyper-responsiveness to the signal. For example, the anti-apoptotic effect of PTH on osteoblasts requires Runx2-driven transcription of survival genes but PTH also induces the Smurf1-mediated proteasomal degradation of this trans-acting protein (Bellido et al., 2003). This mechanism appears to support a self-limiting response of the hormone’s own anabolic action, though it has not been tested in vivo. Similarly, up-regulation of the CREM gene, the products of which act as transcriptional attenuators, may keep the anabolic response to intermittent PTH in check, by restraining PTH-induced osteoclastogenesis (Liu et al., 2007). Elucidation of the osteo-inhibitory mechanisms that are activated by anabolic PTH could yield an attractive target for osteoporosis therapy, either alone or in conjunction with PTH treatment, by essentially providing a means to suppress the “off signal” that accompanies each anabolic activation cycle.
We have potentially identified such a target in a series of experiments conducted over the last 12 years (Alvarez et al., 1997, 1998, 2005; Thunyakitpisal et al., 2001; Shah et al., 2004). Nuclear matrix protein 4/cas interacting zinc finger protein (Nmp4/CIZ) is a trans-acting nucleocytoplasmic shuttling protein that, in osteoblasts, acts as a transcriptional attenuator (Shen et al., 2002; Shah et al., 2004; Morinobu et al., 2005). Nmp4/CIZ suppresses PTH-driven matrix metalloproteinase-13 (MMP-13) gene transcription in MC3T3-E1 osteoblast-like cells (Shah et al., 2004). Similarly BMP2-induced up-regulation of alkaline phosphatase, osteocalcin, and type I collagen are all suppressed in MC3T3-E1 cells over-expressing Nmp4/CIZ (Shen et al., 2002), and BMP2- induced bone formation on adult calvariae in vivo was enhanced by CIZ deficiency (Morinobu et al., 2005). Collectively, our studies indicate that Nmp4/CIZ activation induced by anabolic regimens of PTH might be a mechanism to partially antagonize the osteogenic response of bone tissue to therapeutic doses of PTH. From these data, an obvious question emerges: does the anabolic response to PTH improve when Nmp4/CIZ is disabled/deleted? If so, Nmp4/CIZ might represent an attractive pharmacologic target for inhibition in the search for new osteoporosis therapies.
In the present communication, we examine the role of Nmp4/CIZ in normal skeletal homeostasis and in regulating the response of the skeleton to intermittent PTH. We engineered mice with a loss-of-function mutation in Nmp4/CIZ, and measured bone mass, density, structure, and gene expression, at several time points. We further evaluated the ramifications of the null mutation on anabolic PTH signaling by challenging the mice with intermittent PTH for 7 weeks. Nmp4-null mice exhibited a modest gain in skeletal BMD and BMC between 8 and 16 weeks of age as compared to WT animals. Notably, the Nmp4-knockout (KO) animals displayed a significantly enhanced hormone-induced gain in femoral cancellous bone. The present data and our previous in vitro studies support our hypothesis that Nmp4/CIZ is a key component to an inhibitory mechanism of bone formation. As such, it appears to act as a transcriptional attenuator that suppresses osteoid synthesis thus limiting anabolic PTH-mediated gains in cancellous bone.
Our local Institutional Animal Care and Use Committee (IACUC) approved all experiments and procedures involving the production and use of the experimental mice described in this study. Nmp4-null mice were prepared under our direction at the in Genious Targeting Laboratories, Inc., Stony Brook, NY. The strategy used in preparing these mice involved replacing the region of the Nmp4 gene containing coding exons 4–7 with the Neo gene cassette (see Fig. 1A). Briefly, a 14.5 kb mouse genomic DNA fragment was cloned from the mouse 129Sv/Ev lambda genomic library. This genomic fragment contains exons 4–7 of the mouse Nmp4 gene. The targeting vector was constructed by using 3 kb DNA fragment as the short arm, which was a PCR fragment from primers NMPYA1 to NMPYA2 (Table 1). Primer NMPYA1 is located 3 kb upstream of exon 1 and primer NMPYA2 is located in exon 1 and 45 bp upstream of the ATG start codon. The short arm was inserted into the 5′ end of the Neo gene cassette using the MluI site. The long arm was an 11 kb genomic fragment that starts from BsrGI site to the end of the lambda clone.
Ten micrograms of the targeting vector was linearized by NotI and then transfected by electroporation of iTL1 embryonic stem cells (ES, 129Sv/Ev). After selection in G418 antibiotic, 200 resistant colonies were expanded for PCR analysis to identify recombinant clones. Primers, A1, A2, and A3 (Table 1) were designed upstream 5′ to the short homology arm outside the region used to generate the targeting construct. PCR reactions (94°C 20 sec; 62°C60 sec; 72°C 240 sec; 35 cycles) using A1, A2, or A3 with the N1 primer (Table 1) at the 5′ end of the Neo cassette amplified 3.4, 3.5, and 3.5 kb fragments, respectively (data not shown).
The correctly targeted ES cell lines from 129SvEv ES clones were microinjected into C57BL/6J blastocysts. The chimeric mice were crossbred with the C57BL/6J mice to generate germline transmission. The F1 heterozygotes were identified via PCR screening using the primer pairs A2/N1 and A3/N1 and then re-confirmed by primer pair A1/N1 with negative and positive controls (data not shown).
The progeny of the mating of the first heterozygotes were a hybrid of 129SvEv and C57BL/6J. Both WT and Nmp4-KO mice in a 129SvEv and C57BL/6J F2 background were derived from the original heterozygous cross. In addition to the Nmp4-KO-F2 and Nmp4-WT-F2 mice we further backcrossed the KO mice onto a C57BL/6J background through six generations (Nmp4-KO-N6) and it were these mice that we have compared to C57BL/6J mice obtained from The Jackson Laboratories (Bar Harbor, ME) and used for the hormone studies. The genotypes of our mice were confirmed by two independent methods. Genomic DNA isolated from tail biopsies at weaning was evaluated by PCR analysis. The location of the primers for PCR (WT1, WT2, Neo1, Neo2, Table 1) are indicated in Figure 1A. Primers WT1 and WT2 amplify a 465 bp fragment and the Neo primers amplify a 512 bp fragment (see Fig. 1B). Additionally, Nmp4 expression in primary bone marrow-derived osteoblast cultures from WT and Nmp4-KO mice was compared using quantitative real-time PCR analysis (qRT-PCR) (see below for experimental details).
Areal bone mineral density (aBMD; mg/cm2) and bone mineral content (BMC; g) were measured for the post-cranial skeleton by dual-energy X-ray absorptiometry (DEXA) using an X-ray pixiMUS mouse densitometer (PIXImus II; GE-Lunar Corp., Madison, WI). Mice were anesthetized with isoflurane (2% at 1.5 L/min) and placed in a prone position with limbs outstretched on the pixiMUS platform. Body weight was recorded at this time. Growth rates were determined from the slopes of the weight×age regression lines.
To assess trabecular micro architecture at the distal femoral metaphysis, the distal 25% of each femur was scanned on a desktop microcomputed tomographer (μCT 20, Scanco Medical AG, Bassersdorf, Switzerland) at 9 μm resolution. A microfocus X-ray tube with a focal spot of 10 μm was used as a source. For each slice, 600 projections were taken over 216° (180° plus half of the fan angle on either side). Precisely 205 microtomograph slices were acquired per bone using a slice increment of 17 μm. Distal femur stacks were reconstructed to the 3rd dimension using a standard convolution-backprojection procedure with a Shepp–Logan filter using a threshold value of 270. From the 3D constructs, trabecular bone volume per total volume (BV/TV), trabecular thickness (Tb Th), spacing (Tb Sp), and number (Tb N), connectivity density (Conn D), and structure model index (SMI) were calculated using the Scanco software. To assess cortical architecture, a single slice was taken through the midshaft femur, and the total area (TA), cortical area (CA), and second moments of inertia (I) were calculated from each midshaft slice.
Prior to the start of an experiment (8 weeks of age), mice, female WT and Nmp4-KO mice were given 0.01 ml sterile saline by subcutaneous (sc) injection once daily to acclimatize them to handling. At 10 weeks of age, mice were sorted into four groups of 11–12 mice each based on equivalent mean-group-body weight and were injected with human PTH 1-34 (Bachem Bioscience, Inc., Torrance, CA) at 30 μg/kg/day s.c., daily or vehicle control (0.2% BSA/0.1% 1.0 mN HCl in saline, Abbott Laboratory, North Chicago, IL) for 48 days and sacrificed on Day 49.
The muscle and connective tissue were removed from femurs and tibias of 6- to 8-week-old mice, the bones then briefly rinsed in 70% ethanol, and placed in PBS/HBSS at room temperature (Huang et al., 2004). The distal and proximal ends of the femurs and tibias were then cut and the marrow flushed with a tuberculin syringe, filled with complete α-MEM and cells, passage zero (P0), were typically seeded at 3,000–6,000 cells/mm2. On Day 5, the fresh medium was supplemented with 50 μg/ml of ascorbic acid and subsequently, the medium was replenished three times/week.
To harvest RNA from the femurs of 8-week-old female WT and Nmp4-null mice, freshly excised bones were transferred to liquid nitrogen and ground to a fine powder. The frozen bone powder was further homogenized in Trizol®(Invitrogen, Carlsbad, CA) using a Tissue-Tearor™(BioSpec Products, Inc., Bartlesville, OK). RNA was extracted from the Trizol-bone powder samples with chloroform and precipitated with isopropanol. The RNA pellet was further processed using the RNeasy Plus Mini Kit (QIAGEN, Inc., Valencia, CA). First strand cDNA synthesis was performed with First-Strand cDNA Synthesis Kit (Amersham Bioscience, Piscataway, NJ). Real-time PCR primers and probes were obtained from Assays-on-Demand™ (Applied Biosystems, Foster City, CA) for the following genes: alkaline phosphatase (Mm01187117_m1), COL1A1 (Mm00801666_g1), IGF-1 (Mm0043559_m1), MMP-13 (Mm00439491_m1) OPG (Mm00435452_m1), osteopontin (Mm00436767_m1), osterix (Mm00504574_m1), RANKL (Mm00441908_m1), and Runx2 (Mm00501578_m1). Custom design primer/probes were prepared for osteocalcin and Nmp4, and neomycin phospho transferase (Neo) (Table 1). We used the TaqMan Universal PCR Master Mix (Applied Biosystems) for amplification in a Mastercycler ep realplex2 real-time PCR system (Eppendorf, Westbury, NY). The qRT-PCR amplifications were performed in a sealed 96-well optical plate. The reaction conditions were as follows: 2 min at 50°C; 10 min at 95°C; 15 sec at 95°C; 1 min at 60°C; 40 cycles of 15 sec at 95°C, and 1 min at 60°C. Prior to assessing differences between WT and KO target gene expression we evaluated our experimental preparations for candidate normalizing genes using the Taqman Mouse Endogenous Control Array (Applied Biosystems) and choose RPLP2 (Mm03059047_gH). Samples that had equivalent levels of RPLP2 RNA expression were chosen for analysis. The ΔΔCT method was used to evaluate gene expression between WT and KO animals and the data represent the mean±standard deviation from at least six mice per genotype. Finally, BMSCs were seeded into 12-well culture dishes and at the indicated days post-seeding total cellular RNA was harvested from at least 6 independent cell cultures per genotype for qRT-PCR analysis. We determined that both RPLP2 and GAPDH were suitable as normalizing genes for these samples.
Statistical analysis was computed using JMP Version 7.0.1 (SAS Institute, Cary, NC). Typically, we used a two-factor ANOVA to determine if genotype and age, or genotype and treatment had a significant effect on a particular phenotypic parameter as well as to determine if interaction occurred between the independent variables of gene×age or gene×treatment. If the two-factor ANOVA indicated a significant effect for any of the independent variables, the data were then analyzed with a one-factor ANOVA followed by a Tukey’s HSD post hoc test to determine significant differences between experimental groups. For some experiments, unpaired t-tests were employed as indicated. Genotype frequencies among mouse pups generated from heterozygous parents were tested for deviation from Mendelian ratios using the Chi-square test for goodness-of-fit. Data are presented as mean±SD. Statistical significance was taken at P<0.05.
Homologous recombination was used to replace exons 4–7 with the Neo gene cassette and the F1 heterozygotes were identified as described in Materials and Methods Section (Fig. 1A). The correct recombination event was confirmed both by PCR analysis of mouse tail DNA (Fig. 1B) and by analysis of Nmp4 expression in primary BMSC-derived osteoblasts (Fig. 1C). Animals homozygous for the Nmp4 deletion allele lacked any detectable Nmp4 mRNA in primary osteoblasts derived from bone marrow stromal cells (Fig. 1C), additionally, no Nmp4mRNA was detected in the femurs of Nmp4-KO mice (data not shown). Like the CIZ-deficient mice (Nakamoto et al., 2004; Morinobu et al., 2005), our Nmp4-KO mice appeared to be indistinguishable from WT littermates, were viable to adulthood, and showed no gross skeletal abnormalities (Fig. 1D). There was no significant difference in femur length at 8 weeks (WT=14.30±0.20 mm; KO=14.17±0.40 mm) and 17 weeks (WT=15.35±0.20 mm; KO=15.20±0.40 mm) of age between the Nmp4-KO-N6 and WTC57 BL/6J mice. In the process of backcrossing the Nmp4-KO genotype onto the C57BL/6J background we observed that matings between heterozygous (HZ Nmp4+/−) mice produced a 1.4:2.1:0.5 genotype ratio among pups, which was found to deviate significantly from the 1:2:1 expected Mendelian frequencies (χ2 = 6.1, P<0.05). The diminished yield of KO pups from the predicted Mendelian ratios suggests that some of the KO embryos die either prior to birth or immediately after birth and we are presently investigating this phenomenon. Additionally, we observed that the size of the litters from Nmp4-KO-F2 crosses were modestly smaller than the litters from Nmp4- WT-F2 crosses of mice (pups/WT litters9 litters=7.4±1.4; pups/KO litters10 litters=5.4±2.6, average±SD; Prob> |t| 0.051). These observations are consistent with the sporadic infertility observed in CIZ-deficient mice due to spermatogenic cell degeneration (Nakamoto et al., 2004). Finally, Nmp4-KO-F2 and Nmp4-WT-F2 litters produced on average an equal number of males and females (% males:% females/litter:WT 52.8±23.4:47.2±23.4; KO 44.6±19.8: 55.4±19.8; average±SD).
We observed a modest enhancement of baseline BMD and BMC in the Nmp4 KO mice as measured by DEXA. At 8 weeks of age the Nmp4-KO-N6 skeleton exhibited an approximate 8% increase in BMD in the whole body (WB) skeleton (P<0.05), tibia (P<0.05), and spine compared to the same bones of the WT mice (see Fig. 2A,C,G) whereas the difference in the femur BMD between these mice was less pronounced (Fig. 2E). Similarly, the Nmp4-N6-KO WB skeleton, tibia, spine and femur exhibited on average an approximate 10% enhancement in BMC as compared to their WT counterparts but these differences were not statistically significant (Fig. 2B,D,F,H). These trends were generally maintained at 16 weeks of age and the enhanced femur and spine BMCs achieved statistical significance (Fig. 2). Similarly, the Nmp4- KO-F2 mice exhibited a statistically significant 4% enhancement in WB BMD (P<0.05) and on average a 4% increase in WB BMC between 8 and 13 weeks (data not shown).
Consistent with the small increase in BMD and BMC we observed a modest, near significant enhancement of COL1A1 mRNA expression in the 8-week-old Nmp4-null femurs compared to WT bone using qRT-PCR (Fig. 3). However, no differences in the femur expression of Runx2, osterix, alkaline phosphatase IGF-1, MMP-13, OPG, osteocalcin, osteopontin, or RANKL were detected between WT and KO mice (data not shown). Bone marrow stromal cells from Nmp4-null and WT mice cultured under standard osteogenic conditions for 10–14 days (Huang et al., 2004) showed no difference in basal expression of these genes (data not shown).
The Nmp4 mutant alleles on a pure B6 background yielded a significant, 15% increase in body weight among the KO mice, compared to their WT controls (Fig. 4). Additionally, PIXImus analysis revealed no significant difference in percent body fat between WT and Nmp4-null mice (data not shown). Interestingly, the Nmp4-null mice prepared in a 129SvEv×C57BL/6 F2 background exhibited a significant 4% decrease in body weight as compared to the Nmp4-WT-F2 controls between 8 and 13 weeks of age as well as a modest but significant 2.5% decrease in percent body fat (P<0.05; data not shown). This is similar to the previous observation that CIZ-deficient mice prepared in a DBA×C57BL/6 F2 background exhibited an approximately 10% decrease in body weight as compared to the CIZ-WT-F2 mice at 8 weeks of age (Morinobu et al., 2005). The opposing effects of Nmp4 mutation on body weight in the null-N6 and -F2 mice suggest the existence of a modifier gene(s) influencing this aspect of the mutant phenotype.
To evaluate the influence of Nmp4 on the skeletal response to anabolic doses of human PTH, WT and Nmp4-KO mice were injected daily with PTH or vehicle for 7 weeks. Intermittent doses of PTH had a similar effect on DXA-derived skeletal BMD and BMC in both genotypes, enhancing WB, femur, spine, and tibia BMD and BMC by two- to threefold over vehicle-treated mice in both the WT and Nmp4-null groups (Fig. 5A–H). While the PTH regimen was effective in building bone among both genotypes, we were unable to detect any significant differences between WT and Nmp4 null in responsiveness to PTH using DXA-derived measurements.
Hormone treatment modestly enhanced the growth rates (body weight gain from 10 to 17 weeks) of both the WT and Nmp4-KO mice, but the impact of PTH on weight gain was dramatically less than that observed for bone gain (Fig. 6). The Nmp4-KO mice gained significantly less body mass during the treatment period than their WT counterparts, regardless of treatment, but hormone did not affect weight gain differently within the two genotypes (i.e., no significant interaction term was found).
PTH had a more dramatic impact on enhancing trabecular microarchitecture of the distal metaphyseal femur in the Nmp4-KO mice as compared to the WT mice (Fig. 7A–F). We examined the trabecular parameters of BV/TV, Conn D, SMI, Tb N, Tb Th, and Tb Sp using μCT analysis as described in Materials and Methods Section. We observed both genotype and treatment effects, as well as a genotype×treatment interaction for BV/TV, Conn D, and SMI (Fig. 7A–C). WT and Nmp4-null mice under the vehicle-control regimen exhibited equivalent BV/TV, Conn D, and SMI. Although hormone treatment significantly altered these parameters in both types of mice the magnitude of PTH-induced changes in the Nmp4-null mice ranged from 2.3- to 3.5-fold greater than the changes observed in the WT animals (Fig. 7A–C). Similarly, both the vehicle-treated WT and Nmp4-KO mice exhibited equivalent Tb N and Tb Th indices but the Nmp4-null mice response to hormone was approximately 1.5-fold greater than that of the WT mice in regard to these parameters (Fig. 7D,E). Finally, the vehicle-treated KO mice exhibited a significantly smaller Tb Sp than the vehicle-treated WT animals but the observed PTH-induced decrease in Tb Sp was similar in magnitude in both genotypes (Fig. 7F). Typical μCT scans from each of the four treatment groups is shown in Figure 8.
Next we examined the cortical bone geometry of the midshaft femurs from our four experimental groups using μCT analysis. The maximum and polar moments of inertia (IMAX and J) were statistically identical (see Fig. 9A) and revealed only a modest trend toward enhanced bending and torsional properties among the KO femurs when compared to the corresponding WT femurs. IMIN values for the KO VEH and KO PTH experimental groups were significantly greater than those values obtained from the WT VEH and WT PTH groups, respectively (P<0.05). Hormone significantly enhanced these parameters in both WT and null mice (Fig. 9A), but did so similarly in both genotypes (i.e., no genotype×treatment interaction). CA and TA have very similar profiles to those of the moments of inertia data (Fig. 9B), but MA was not influenced by either genotype or treatment (data not shown). Hormone significantly enhanced CA in both the WT and null mice but only significantly increased TA in the WT mice (Fig. 9B). Strikingly, the values of IMAX, IMIN, J, CA, and TA for the PTH-treated WT mice were not significantly different from those values observed for the untreated mice harboring Nmp4-null alleles.
Nmp4/CIZ was independently discovered as a PTH-responsive osteoblast nuclear matrix DNA-binding protein (Nmp4, Alvarez et al., 1998) and as a nucleocytoplasmic shuttling transcription factor that binds to p130cas in rodent fibroblasts (CIZ, Nakamoto et al., 2000). Although ubiquitously expressed, its activity appears to be particularly significant to skeletal homeostasis.
Nmp4/CIZ acts to moderately suppress the acquisition of bone during skeletal growth and maturation. We observed an 8% and 10% increase in BMD and BMC, respectively throughout the 8- and 16-week-old Nmp4-null skeleton and this is similar to the previous observation that 8-week-old CIZ-deficient mice exhibited an enhanced femoral radiopacity and 40% increase in femoral BV/TV (Morinobu et al., 2005). The enhanced acquisition of bone in CIZ-deficient animals resulted from elevated bone formation and mineral apposition rates and not to any perceptible change in bone resorption parameters (Morinobu et al., 2005). This is consistent with our observation that the baseline expression of COL1A1 mRNA was modestly elevated in the femurs of Nmp4-KO mice as compared to their WT counterparts. Furthermore, we have previously demonstrated that Nmp4 binds to the regulatory elements of rat COL1A1 and that over-expression of Nmp4 suppresses COL1A1 expression in rat osteoblast-like cells (Alvarez et al., 1997, 1998; Thunyakitpisal et al., 2001). Nevertheless, basal expression of selected genes supporting bone cell phenotype is indistinguishable in cultures of WT and Nmp4-KO mature osteoblasts. Whether there are differences in gene expression profiles between WT and Nmp4-null bone cells at earlier or later stages of differentiation is currently under investigation.
The additional effect of Nmp4/CIZ on body weight raises the question as to whether it plays a role in regulating the lineage fate of mesenchymal stem cells. Although both the F2- and N6- null mice exhibited an increase in BMD and BMC, this was not the case for body weight and percent body fat, suggesting that modifier genes influence Nmp4/CIZ regulation of some aspects of body composition. The Nmp4-KO-F2 mice exhibited a modest (4%) but significant decrease in body weight similar to that reported for the CIZ-KO-F2 mice (Morinobu et al., 2005). Our Nmp4-KO-F2 mice also showed a small but significant 2.5% reduction in percent body fat. This increase in bone and decrease in fat in the F2-null mice is consistent with the putative inverse relationship between the commitment of bone marrow-derived mesenchymal stem cells to the adipocyte and osteoblast lineage pathways (Gimble et al., 2006 and references therein). However our Nmp4-KO-N6 mice were on average 15% heavier than the WT counterparts but exhibited no difference in percent body fat suggesting an uncoupling of Nmp4/CIZ action on adipogenesis and osteogenesis in the pure B6 background. We are presently conducting a more comprehensive investigation on Nmp4/CIZ regulation of body composition.
The most profound impact of the loss-of-function Nmp4 mutation in our mice is the enhanced PTH-induced acquisition of trabecular bone without significantly influencing hormone-stimulated gains in cortical bone. The microarchitecture of the distal femur trabecular compartments of the vehicle-treated Nmp4-KO and WT mice at 17 weeks of age were identical as measured by BV/TV, Conn D, SMI, Tb N, and Tb Th but the PTH-induced changes in these parameters were between 1.5- and 3.5-fold greater in the Nmp4-null mice. In other words, the removal of Nmp4-KO facilitated greater PTH-induced trabecular bone gain. However, the impact of anabolic PTH on skeletal BMD and BMC were similar in both Nmp4-null and WT mice, which was comparable to the hormone response of WT C57BL/6 mice described in a previous study (Iida-Klein et al., 2002). Additionally, PTH-induced increases in the cortical area of the midshaft femur were not significantly different in the Nmp4-KO and WT mice suggesting that Nmp4/CIZ is critical for attenuating hormone-mediated gains in cancellous bone but does not actively suppress PTH-stimulated gains in all skeletal compartments including cortical bone. This is not surprising since the osteoblast and osteoclast responses to PTH are influenced by the apparent distinct microenvironments of cancellous and cortical bone. For example, transgenic mice expressing a constitutively active parathyroid hormone/parathyroid hormone-related protein receptor in osteoblast cells exhibited a significant increase in trabecular bone volume and a decrease in cortical thickness (Calvi et al., 2001). Similarly, PTH-stimulated periosteal bone formation was twofold higher in estrogen-replete female beta-arrestin2-null mice as compared to their WT counterparts, but the hormone response of the femoral and vertebral cancellous compartments were not as appreciably different in these two mice (Bouxsein et al., 2005).
Our previous in vitro studies suggest that Nmp4/CIZ suppresses PTH-induced gains in bone by acting as a transcriptional attenuator (Thunyakitpisal et al., 2001; Shah et al., 2004). Over-expression of Nmp4/CIZ in UMR-106-01 rat osteoblast-like cells not only suppresses basal COL1A1 transcription (Thunyakitpisal et al., 2001) but also PTH induction of MMP-13 (Shah et al., 2004). There are conserved Nmp4/CIZ cis-elements in the human, rat, and mouse PTH response region of the MMP-13 gene (Bidwell et al., 2001; Shah et al., 2004). In an earlier study, we prepared a series of promoter–reporter constructs of the MMP-13 gene containing the first 1,329 nucleotides of the 5′-regulatory region and introduced null-binding mutations in the Nmp4/CIZ element and/or the proximal Runx2 cis-element (Shah et al., 2004). The activity of the WT MMP-13 construct increased by twofold when challenged with rat PTH (1–34) for 6 h in UMR-106-01 rat osteoblast-like cells. However, the promoter–reporter construct harboring a null-binding mutation in the Nmp4/CIZ site responded to hormone stimulation with a significantly enhanced fivefold increase in activity. Finally, mutation of a proximal Runx2 site within this PTH region slightly attenuated the PTH response, however, mutation of this site and the Nmp4/CIZ element together resulted in an 11-fold increase in PTH-induced activity (Shah et al., 2004). Therefore, as a trans-acting protein Nmp4/CIZ attenuates the magnitude of the transcriptional response to PTH and its proximity to Runx2 may enhance this function. Furthermore, CIZ over-expression suppressed BMP2-induced Runx2 promoter activity in MC3T3-E1 osteoblast-like cells (Shen et al., 2002) thus Nmp4/CIZ may govern the action of Runx2 not only by interfering with its activity along the regulatory regions of PTH target genes, but also by attenuating its expression, although we did not observed an enhanced baseline expression of Runx2 in the femurs of our Nmp4-KO mice or from osteoblasts derived from these bones. Consistent with our hypothesis that the Nmp4/CIZ pathway may be linked to an endocrine feedback circuit, we have reported that PTH attenuated the activities of the mouse Nmp4 P1 and P2 promoters in MC3T3-E1 osteoblasts (Alvarez et al., 2005) thus providing a potential means for the hormone to regulate its own regulator. Nevertheless further studies are required to elucidate the cellular and molecular events responsible for the enhanced response to anabolic doses of PTH in the Nmp4-null mice.
Disabling Nmp4/CIZ appears to enhance anabolic gains in bone and neutralize catabolic bone loss. As previously mentioned CIZ attenuates BMP2-mediated increases in osteoblast gene expression in vitro (Shen et al., 2002) and bone formation in vivo (Morinobu et al., 2005) and the mutations in the MMP-13 Nmp4/CIZ cis-element described above also enhance this gene’s response to prostaglandin E2 (Shah et al., 2004). Remarkably CIZ deficiency also abrogates unloading-induced bone loss (Hino et al., 2007). Those data suggest that Nmp4/CIZ might constitutively suppress osteoid synthesis and neutralizing its action allows matrix production under typically catabolic conditions. The suppressive action of Nmp4/CIZ on osteoid synthesis may be particularly sensitive to changes in the mechanical environment of the osteoblast since both PTH and load have a physical impact on bone cells (Egan et al., 1991; Horikawa et al., 2000) and these agonists activate some of the same signaling pathways in bone cells (Ryder and Duncan, 2001; Zhang et al., 2006). Consistent with the absence of bone loss in unloaded CIZ-deficient mice, Nmp4/CIZ contributes to fluid shear stress-induced changes in MMP-13 expression in MC3T3-E1 cells via the PTH response region of this gene (Charoonpatrapong-Panyayong et al., 2007).
Nmp4/CIZ has been put forward as a potential drug target for the treatment of osteoporosis (Krane, 2005) and the present data strengthen this proposition. Furthermore, its presence in the cytoplasm may make it a more accessible target than the typical transcription factor. In the present study hormone treatment caused a significant decrease in SMI in both the WT and KO mice, indicating that PTH altered trabecular architecture from a rod-like to a plate-like morphology, consistent with an increase in bone strength. Nevertheless, this decrease in SMI was significantly exaggerated in the KO mice (3.5-fold). Additionally, our untreated Nmp4-KO mice exhibited similar bone mass and geometric properties as the PTH-treated WT mice suggesting that therapies designed to inhibit Nmp4/CIZ might yield similar benefits to bone mass and strength as those achieved via daily PTH administration.
Contract grant sponsor: NIH NIDDK;
Contract grant number: DK053796.
This work was supported by a grant from NIH NIDDK, contract grant number: DK053796 (to JPB). We thank Marta Alvarez and Rita Shah for their technical assistance.
A. Robling and P. Childress contributed equally to this manuscript.