Hairy and Enhancer of Split-related with YRPW Motif (HEY)2 Regulates Bone Remodeling in Mice*

Background: Notch regulates bone mass and induces Hairy and Enhancer of Split-related with YRPW motif (HEY) in osteoblasts, but the skeletal function of individual HEY proteins is unclear. Results: In male mice, Hey2 inactivation increases bone mass, whereas HEY2 overexpression enhances bone resorption, reducing bone mass. Conclusion: HEY2 regulates skeletal remodeling in male mice, decreasing bone mass. Significance: HEY2 mediates selected skeletal effects of Notch. Notch induces Hairy and Enhancer of Split-related with YRPW motif (Hey)1, Hey2, and HeyL expression in osteoblasts, but the contributions of these genes to the skeletal effects of Notch are not fully understood. HEY1 misexpression has limited skeletal impact, female HeyL null mice display increased bone mass, and Hey2 inactivation is developmentally lethal. To inactivate Hey2 in immature or mature osteoblasts, Hey2loxP/loxP mice were crossed with transgenics expressing CRE under the control of the osterix (Osx-Cre) or osteocalcin (Oc-Cre) promoters to generate Osx-Cre+/−;Hey2Δ/Δ or Oc-Cre+/−;Hey2Δ/Δ mice. Trabecular bone volume increased in 3-month-old Osx-Cre+/−;Hey2Δ/Δ and Oc-Cre+/−;Hey2Δ/Δ male mice and in 1-month-old Oc-Cre+/−;Hey2Δ/Δ female mice, although 3-month-old Oc-Cre+/−;Hey2Δ/Δ females developed osteopenia. Alkaline phosphatase liver/bone/kidney (ALPL) expression and activity were suppressed in osteoblasts from Oc-Cre+/−;Hey2Δ/Δ mice of both sexes. To overexpress HEY2 in osteoblasts, transgenic mice where a 3.6-kb fragment of the rat collagen type-I α1 promoter directs HEY2 expression were created. Three-month-old Hey2 transgenic males exhibited decreased osteoblast activity and increased bone resorption and developed osteopenia at 6 months of age. Hey2 transgenic females exhibited reduced osteoblast number and function, but no changes in bone resorption. HEY2 overexpression in osteoblasts from mice of both sexes inhibited ALPL expression and activity and suppressed osteocalcin transcripts in cells from male mice only. HEY2 overexpression in osteoblasts from male mice enhanced bone resorption by co-cultured splenocytes and induced interleukin-6, a molecule that promotes osteoclastogenesis. In conclusion, HEY2 decreases skeletal mass and regulates bone remodeling in male mice.

Hairy and Enhancer of Split (HES) 2 -related with YRPW motif (HEY) genes encode 3 helix-loop-helix transcription factors termed HEY1, HEY2, and HEY-Like (HEYL) (1)(2)(3). HEY proteins display structural similarities with HES transcription factors and are targets of canonical Notch signaling, a critical regulator of cell differentiation during development and postnatal life (4). HEY proteins play an important role in cardiovascular development. Inactivation of Hey2 results in embryonic lethality due to cardiovascular defects, and the dual inactivation of Hey1 and Hey2 phenocopies the global deletion of Notch1. Similarly, the deletion of Hey1 and HeyL impairs cardiovascular development in mice (5)(6)(7)(8)(9).
The continuous renewal of skeletal tissue is carried out in basic multicellular units. There, osteoclasts resorb bone, and following a reversal phase, new bone matrix is deposited by osteoblasts. Osteoblasts are derived from multipotent mesenchymal stem cells that can differentiate toward the osteoblastic, chondrocytic, or adipocytic lineages (10). The commitment of mesenchymal cells to the osteoblastic fate is controlled by a signaling network that includes bone morphogenetic proteins, Wnt, and Notch (11)(12)(13)(14)(15)(16). Osteoclasts are multinucleated cells formed by fusion and osteoclastic differentiation of mononuclear cell precursors, a process that requires macrophage-colony stimulating factor (M-CSF) and the receptor activator of nuclear factor-b ligand (RANKL). RANK is expressed by osteoclast precursors and is activated following contact with cells expressing the membrane-bound RANKL. The activity of RANKL is opposed by osteoprotegerin (OPG), a soluble RANKL decoy receptor, and the ratio of RANKL over OPG regulates osteoclastogenesis (17,18).
Notch is a transmembrane receptor activated by direct contact with Notch ligands. In the canonical signaling pathway, the Notch intracellular domain is released following a series of cleavages and forms a complex with Epstein-Barr virus latency C promoter binding factor 1, Suppressor of Hairless and Lag-1 (CSL), also termed Rbpj in mice, and with mastermind-like (MAML). The effects of Notch in the skeleton appear to be mediated by the canonical signaling pathway, but the genes responsible for the biology of Notch in bone have not been defined (19,20). Hes1, Hes5, Hey1, Hey2, and HeyL are targets of the canonical Notch signaling pathway in skeletal cells, and as such should account, singly or in combination, for the effects of Notch in the skeleton (21). Previously, we reported that HES1 causes osteopenia by inhibiting bone formation and inducing bone resorption in mice (22). These effects of HES1 phenocopied only partially the skeletal phenotype of mice misexpressing Notch in the skeleton, suggesting that HEY proteins may be downstream effectors of Notch signaling in the skeleton (22). Transgenic expression of HEY1 under the control of the ubiquitously expressed ␤-actin promoter as well as the global inactivation of Hey1 caused mild to modest osteopenia, and global HeyL null mice display increased bone mass (23,24).
In this study, we examined the function of HEY2 in the postnatal skeleton. For this purpose, the skeletal phenotype of mice misexpressing HEY2 was investigated by microcomputed tomography (CT) and by histomorphometric analysis. To understand the cellular mechanisms involved, the differentiation and function of osteoblasts misexpressing HEY2 were examined in vitro.

EXPERIMENTAL PROCEDURES
Hey2 Conditional Null Mice-To study the skeletal consequences of Hey2 inactivation in cells of the osteoblastic lineage, mice where the Hey2 sequence comprised between exon 1 and exon 4 was flanked by loxP sites in a 129Sv/C57BL/6 background, were provided by E. N. Olson (University of Texas Southwestern Medical Center, Dallas, TX) (6). To express CRE recombinase at early stages of osteoblastic differentiation, we obtained C57BL/6 mice where the CRE coding sequence is cloned downstream of an osterix (Osx) promoter (Osx-Cre) (The Jackson Laboratory) (30). In these mice, tetracycline suppresses the Osx promoter activity by virtue of a Tet-Off cassette (31). To express CRE preferentially in mature osteoblasts, mice where a 3.9-kb fragment of the human osteocalcin promoter directs CRE expression (Oc-Cre) were obtained from T. Clemens (Johns Hopkins Medicine, Baltimore, MD) and backcrossed for seven generations into a C57BL/6 genetic background. Osx-Cre or Oc-Cre transgenics were crossed with Hey2 loxP/loxP mice to create Osx-Cre ϩ/Ϫ or Oc-Cre ϩ/Ϫ ;Hey2 loxP/wt mice, which were mated with Hey2 loxP/loxP to obtain Osx-Cre ϩ/Ϫ or Oc-Cre ϩ/Ϫ ;Hey2 loxP/loxP mice. The latter were crossed with Hey2 loxP/loxP to generate an experimental cohort, in which CRE excises the loxP-flanked sequences from the Hey2 loxP allele (Osx-Cre ϩ/Ϫ or Oc-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ ) and littermate controls (Hey2 loxP/loxP ). To prevent Osx-Cre expression during embry-onic development, pregnant mothers were administered chow containing 625 mg/kg of doxycycline (Harlan Laboratories, Indianapolis, IN) from the time of conception to delivery. The presence of the Osx-Cre and Oc-Cre transgenes and of the Hey2 loxP allele was determined by PCR in tail DNA extracts in newborns and adult mice, and primers specific for fatty acidbinding protein 1 (Fabp1) were used as positive controls in the PCR reactions. Recombination of sequences flanked by loxP sites was assessed by PCR in DNA extracts from parietal bone, using primers specific for the Hey2 ⌬ allele. All animal experiments were approved by the Animal Care and Use Committee of Saint Francis Hospital and Medical Center.
Col3.6-Hey2 Transgenic Mice-For preferential expression of HEY2 in osteoblasts, a 1047-bp DNA fragment coding for murine HEY2 (American Type Culture Collection; ATCC, Manassas, VA), preceded by a Kozak consensus sequence, was cloned downstream of a 3.6-kb fragment of the rat Col1a1 (collagen type I ␣1) promoter and upstream of the bovine growth hormone polyadenylation signal (32). Microinjection of linearized DNA into pronuclei of fertilized oocytes from Friend leukemia virus strain B (FVB) mice (Charles River Laboratories, Wilmington, MA) and transfer of microinjected embryos into pseudopregnant mice were carried out at the Gene Targeting and Transgenic Facility of the University of Connecticut Health Center (Farmington, CT). Positive founders were identified by Southern blot analysis of tail DNA and bred to wild type FVB mice to create Col3.6-Hey2 transgenic lines (33). To assess the effects of HEY2 overexpression, heterozygous Col3.6-Hey2 mice were mated to wild type FVB mice to generate heterozygous Col3.6-Hey2 transgenic mice and wild type littermate controls. The presence of the Col3.6-Hey2 transgene was documented by PCR in tail DNA. To assess mRNA expression in skeletal cells, calvariae were frozen in liquid nitrogen at the time of harvest and transferred to Ϫ80°C for storage before RNA extraction. To determine levels of bone resorption, fasting serum concentration of collagen type I C-terminal telopeptide (CTX-I), was measured by using the RatLaps enzyme-linked immunosorbent assay in accordance with the manufacturer's instructions (Immuno Diagnostic Systems, Scottsdale, AZ) (34).
Microcomputed Tomography-Femurs were scanned in 70% ethanol at an energy level of 55 kVp, an intensity of 145 A, and an integration time of 200 ms on a CT 40 scanner (Scanco Medical AG, Bassersdorf, Switzerland). Trabecular bone volume fraction and microarchitecture were evaluated starting ϳ1.0 mm proximal to the femoral condyles. A total of 160 consecutive slices acquired at an isotropic voxel size of 216 m 3 and a slice thickness of 6 m were chosen for analysis. Contours were manually drawn every 10 slices a few voxels away from the endocortical boundary to define the region of interest for analysis. The contours of the remaining slices were iterated automatically. Trabecular regions were assessed for bone volume fraction, trabecular thickness, number and separation, connectivity density, and structure model index (SMI), using a Gaussian filter ( ϭ 0.8) and a user-defined threshold (35). A total of 100 slices for the cortical region were measured at the middiaphysis of each femur with an isotropic voxel size of 216 m 3 and a slice thickness of 6 m. For mid-diaphysis analysis, con-tours were iterated across the 100 slices along the cortical shell, excluding the bone marrow cavity. Analysis for cortical thickness was performed using a Gaussian filter ( ϭ 0.8) and a user-defined threshold (35).
Bone Histomorphometric Analysis-Static and dynamic histomorphometry of femurs was carried out after injection with 20 mg/kg calcein and 50 mg/kg demeclocycline, at an interval of 2 days for 1-month-old mice and 7 days for 3-and 6-month-old mice. Animals were sacrificed by CO 2 inhalation 2 days after the demeclocycline injection. Femurs were sectioned on a microtome at a thickness of 5 m (Microm, Richards-Allan Scientific, Kalamazoo, MI) and stained with 0.1% toluidine blue. Static parameters of bone formation and resorption were measured in a defined area between 360 and 2160 m from the growth plate, using an OsteoMeasure morphometry system (Osteometrics, Atlanta, GA) (36). For dynamic histomorphometry, mineralizing surface per bone surface and mineral apposition rate were measured on unstained sections under ultraviolet light, using a triple diamino-2-phenylindole fluorescein set long pass filter, and bone formation rate was calculated. The terminology and units used are those recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (37).
Alkaline Phosphatase Activity-Alkaline phosphatase activity was determined in cell extracts by the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol, measured by spectroscopy at 405 nm according to manufacturer's instructions (Sigma-Aldrich). Data are expressed as nanomoles of p-nitrophenol released per minute per g of protein measured by the DC protein assay (Bio-Rad Laboratories).
Osteoblast-Splenocyte Co-Cultures and Pit Formation Assay-Primary osteoblasts from male Col3.6-Hey2 transgenic mice and male littermate controls were seeded on BioCoat discs (BD Biosciences), and after reaching confluence, cultured in the presence of 100 g/ml ascorbic acid and 5 mM ␤-glycerophosphate. Primary splenocytes were harvested from spleens aseptically removed from 1-month-old wild type FVB male mice, and 1 ϫ 10 6 cells/cm 2 were seeded on the layer of primary osteoblasts in the presence of 10 nM 1,25 dihydroxyvitamin D 3 (BioMol International, Plymouth Meeting, PA) or phosphatebuffered saline, as control (44 -46). Splenocytes and primary osteoblasts were cultured for 7 days, cells were removed with bleach for 5 min, and BioCoat discs were stained with von Kossa. Stained discs were photographed on a white light background and a digital grayscale image analyzed with Adobe Photoshop (Adobe Systems, Inc., San Jose, CA). The negative image was obtained with the invert feature of Adobe Photoshop, brightness and contrast were maximized, and the area of resorption was calculated as the percentage of pixels contained in half of the grayscale, determined by using the histogram function of Adobe Photoshop (22).
Statistical Analysis-Data are expressed as means Ϯ S.E. Statistical differences were determined by Student's t test or anal-ysis of variance with Schaeffe's post hoc analysis for pairwise or multiple comparisons, respectively (47).

RESULTS
Hey2 Inactivation in Cells of the Osteoblastic Lineage Causes a Transient Increase in Bone Volume-To investigate the function of Hey2 at early stages of osteoblast maturation and in differentiated osteoblasts, the skeletal phenotype of Osx-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ or Oc-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ mice was compared with the phenotype of littermate Hey2 loxP/loxP controls of the same sex. Recombination of Hey2 sequences flanked by loxP sites was detected in DNA extracts from calvariae of 1-and 3-month-old Osx-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ and Oc-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ mice, whereas recombination was not observed in littermate controls (Fig. 1, A and B, and data not shown). Osx-Cre ϩ/Ϫ ; Hey2 ⌬/⌬ and Oc-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ mice were viable, appeared normal, and did not exhibit differences in mortality when compared with littermate controls.
In initial experiments, we demonstrated that Oc-Cre, Osx-Cre, and Hey2 loxP/loxP did not exhibit an appreciable skeletal phenotype as determined by CT at 1 month of age (48). One-month-old Osx-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ male mice did not display a skeletal phenotype. CT analysis of femurs from 3-month-old Osx-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ male mice revealed increased trabecular bone volume and connectivity, secondary to increased trabecular number (Table 1, Fig. 1C). However, no changes in parameters of bone formation or bone resorption were observed by histomorphometric analysis (data not shown). No skeletal phenotype was observed in Osx-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ female mice by CT analysis (Table 1, Fig. 1C), indicating that Hey2 inactivation at early stages of osteoblast differentiation increases bone mass only in male mice.
Oc-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ male mice at 1 month of age did not exhibit changes in trabecular bone volume, although these mice displayed a limited reduction in mineral apposition rate and bone formation rate (Fig. 1D, Table 2). Three-month-old Oc-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ male mice developed increased bone volume/tissue volume, indicating that the suppressed osteoblast function at 1 month of age does not translate into osteopenia in older mice (Table 2, Fig. 1D). In addition, inactivation of Hey2 in 3-month-old male mice resulted in lower SMI (  Recombination of the Hey2 loxP allele was determined by PCR. Control reactions conducted in tail DNA from female Hey2 loxP/loxP mouse or water were performed to confirm specificity of the primers. All reaction products were separated by electrophoresis on the same gel, and results from representative individuals are shown. C and D display microcomputed tomography images of proximal trabecular bone and cortical bone at the midshaft of femurs from representative 3-month-old male or female Osx-Cre ϩ/Ϫ (C) or Oc-Cre ϩ/Ϫ (D); Hey2 ⌬/⌬ mice; (Hey2 null) or littermate Hey2 loxP/loxP controls of the same sex (Control).
1D), indicative of a higher ratio of plate-like over rod-like trabeculae (49). A modest increase in cortical thickness was observed (Table 2, Fig. 1D). Female Oc-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ mice did not exhibit a skeletal phenotype at 1 month of age except for a modest reduction in mineral apposition rate, and at 3 months of age, they exhibited modest osteopenia by CT, but not by histomorphometry (  Fig. 1D). The skeletal phenotypes of Osx-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ and Oc-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ mice were transient, and at 6 months of age, these mice were not different from littermate Hey2 loxP/loxP controls, as determined by CT (not shown).
Hey2 Inactivation Impairs Osteoblast Function in Vitro-To explore the cellular mechanisms that lead to suppressed bone  formation in Oc-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ mice, primary osteoblastic cells were harvested from Oc-Cre ϩ/Ϫ ;Hey2 ⌬/⌬ male or female mice and littermate Hey2 loxP/loxP controls of the same sex. Alkaline phosphatase transcripts and activity were decreased in the context of Hey2 inactivation in primary calvarial osteoblasts from mice of both sexes (Fig. 2). These findings are in agreement with the decrease in mineral apposition rate exhibited by Hey2 null mice and indicate that HEY2 is required for full osteoblastic function.

HEY2 Overexpression in Osteoblasts Uncouples Bone Formation from Bone
Resorption-To test further the function of HEY2 in the skeleton, the effects of the preferential HEY2 overexpression in osteoblasts were investigated in Col3.6-Hey2 transgenic mice at 1, 3, and 6 months of age. Three transgenic founders were obtained, and only one male founder mouse transmitted the transgene to the offspring, allowing the establishment of a Hey2 transgenic line. Col3.6-Hey2 transgenics were born at the expected Mendelian ratio and appeared normal and healthy. qRT-PCR analysis demonstrated that Hey2 mRNA levels, corrected for Rpl38 expression, in calvariae from 1-month-old Col3.6-Hey2 transgenic males and females mice were increased 32.9 Ϯ 3.4-and 32.2 Ϯ 10.6-fold (p Ͻ 0.05), respectively, in comparison with control littermates of the same sex, confirming expression of the Col3.6-Hey2 transgene. CT revealed that Col3.6-Hey2 male transgenics did not exhibit a phenotype at 1 or 3 months of age, but were osteopenic at 6 months of age. The SMI value closer to 3 in the Col3.6-Hey2 transgenic male mice indicated a preponderance of rod-like over plate-like trabeculae, suggesting that HEY2 affects bone microarchitecture (Table 3, Fig. 3) (49). Histomorphometric analysis revealed that Col3.6-Hey2 transgenics had increased osteoclast number and eroded surface and decreased bone formation rate at 3 months of age (Table 3). However, HEY2 overexpression did not affect serum levels of CTX-I, a marker of bone resorption, in 1-month-old male mice (data not shown) (34). The changes in the cellular parameters observed at 3 months of age may explain the reduced bone volume observed in 6-month-old male mice, suggesting that overexpression of HEY2 in osteoblasts causes osteopenia due to an uncoupling of osteoblast and osteoclast activities. CT and histomorphometric analysis indicated that Col3.6-Hey2 transgenic female mice did not exhibit an obvious skeletal phenotype at 1, 3, and 6 months of age, except for a decrease in bone formation rate at 6 months of age (Table 4, Fig. 3). No changes in osteoclast number, eroded surface, or serum levels of CTX-I were observed, indicating that HEY2 overexpression  (Hey2 null, black bars), or littermate Hey2 loxP/loxP controls of the same sex (Control, white bars) and cultured under conditions favoring osteoblastogenesis. Total RNA was extracted, and mRNA was reverse-transcribed and amplified by qRT-PCR in the presence of specific primers. Data are expressed as Alpl copy number, corrected for Rpl38 copy number. Values are means Ϯ S.E., n ϭ 3-4. Alkaline phosphatase activity was determined in cells extracted with Triton X-100, and data are expressed as nanomoles of p-nitrophenol/min/g of total protein. Values are means Ϯ S.E., n ϭ 6. ϩ, significantly different between day 7 and day 0, p Ͻ 0.05. *, significantly different between HEY2 and control, p Ͻ 0.05.  in osteoblasts regulates osteoclast differentiation and function only in male mice.

HEY2 Overexpression Impairs Osteoblast Function in Vitro-
To understand the cellular effects caused by HEY2 overexpression, osteoblast-enriched cells were harvested from calvariae of Hey2 transgenic male or female mice and littermate controls of the same sex. Increased HEY2 transcripts were confirmed by qRT-PCR in osteoblasts from Col3.6-Hey2 transgenics over a 14-day culture period (Fig. 4). Alkaline phosphatase transcripts and activity were suppressed by HEY2 in cells from both sexes (Fig. 4). Osteocalcin expression was detected only at 7 and 14 days of culture and was suppressed in osteoblasts from Col3.6-Hey2 male but not female transgenic mice (Fig. 4). These findings confirm that HEY2 overexpression impairs osteoblast differentiation and function in both sexes and could explain the decreased bone formation and osteoblast number observed in vivo.
HEY2 Overexpression Enhances Osteoclast Function in Vitro-To determine whether the overexpression of HEY2 regulated osteoclast differentiation or function, calvariae from male Col3.6-Hey2 transgenics and littermate wild type controls were co-cultured with wild type FVB splenocytes, a source of mononu-clear osteoclast precursors. Treatment with 1,25-dihydroxyvitamin D 3 induced resorptive activity of wild type splenocytes, and in agreement with the increased eroded surface and osteoclast number observed in the Col3.6-Hey2 transgenic males, osteoblasts from Col3.6-Hey2 transgenics enhanced resorption (Fig. 5A) (44). To investigate possible mechanisms for the stimulatory effect of HEY2 on bone resorption, interleukin-6 (IL6) Rankl and Opg mRNA levels were determined (50). An increase in IL6 transcripts was observed only in male Col3.6-Hey2 transgenic osteoblasts (Fig.  5B), suggesting that HEY2 increases bone resorption by inducing the expression of IL6. HEY2 caused a modest and unexplained increase in Rankl transcripts in osteoblasts from female mice. In accordance with the effects of Notch on OPG expression, HEY2 induced Opg mRNA levels in cells from male mice (Fig. 5B) (26,48). This may represent an indirect compensatory mechanism to temper the effects of HEY2 on osteoclastogenesis.

DISCUSSION
In this study, we investigated the skeletal function of Hey2, a Notch target gene. Hey2 inactivation in cells of the osteoblastic lineage caused an increase in trabecular bone volume, an effect that is consistent with the osteopenic phenotype caused by the activation of Notch signaling in these cells (16,48). The skeletal phenotype was mild, transient, and more pronounced in male than in female mice, indicating that Hey2 plays a modest role in skeletal homeostasis. Inactivation of Hey2 at early stages of osteoblastic differentiation did not affect the number or function of skeletal cells, and a developmental nature for the increased bone mass is excluded because the activity of the Osx promoter was suppressed by doxycycline during embryonic development (30). Global HeyL null mice carrying a heterozy-gous null mutation of Hey1 exhibit increased bone mass, indicating that Hey1, HeyL, and Hey2 have similar functions in the skeleton (24). It is plausible that Hey1 and HeyL compensate for the loss of Hey2 function, preventing detection of subtle changes in osteoblast and osteoclast number and activity, which lead to a modest increase in bone mass. Compensation by Hey1 might explain the sexually dimorphic skeletal phenotype of Hey2 inactivation in immature osteoblastic cells because Hey1 transcript levels significantly increased 1.4-fold in parietal bones when Hey2 was inactivated in female mice, but not in   (Hey2, black bars), or littermate wild type controls of the same sex (Wild Type, white bars) and cultured under conditions favoring osteoblastogenesis. In A, osteoblast-enriched cells from male mice were co-cultured with splenocytes from wild type mice of the same sex in the presence of 10 nM 1,25-dihydroxyvitamin D 3 (Vit.D) or vehicle. After 7 days, cells were removed with bleach, the culture substrate was stained by Von Kossa, and digital pictures were acquired for the estimation of the resorbed area. Data are expressed as the percentage of the resorbed area. Values are means Ϯ S.E., n ϭ 4 -6. *, significantly different between HEY2 and wild type, p Ͻ 0.05. ϩ, significantly different between 1,25-dihydroxyvitamin D 3 and vehicle, p Ͻ 0.05. In B, total RNA was extracted from osteoblast-enriched cell cultures at confluence, and mRNA was reversetranscribed and amplified by qRT-PCR in the presence of specific primers. Data are expressed as Il6, Rankl, and Opg copy number, corrected for Gapdh copy number. Values are means Ϯ S.E., n ϭ 4. *, significantly different between HEY2 and wild type, p Ͻ 0.05. male mice (data not shown). Inactivation of Hey2 in osteoblasts did not affect serum markers of bone remodeling and transiently inhibited bone formation, but the effect was modest, confirming that Hey2 has a limited impact on skeletal function.
Although three Hey2 transgenic founders were obtained, only one transgenic line was established, indicating that excessive levels of HEY2 in cells expressing the 3.6-kb fragment of the Col1a1 promoter are detrimental for embryonic development. The activity of this promoter fragment is not restricted to osteoblastic cells, and expression of HEY2 in nonskeletal tissues may have prevented transmission of the transgene (51,52). Despite these limitations, the data indicate that HEY2 overexpression affects bone remodeling and uncouples bone formation from bone resorption by increasing osteoclastogenesis in male mice and by suppressing osteoblast number and function in both genders. In accordance with the effects of the Hey2 inactivation in osteoblastic cells, Col3.6-Hey2 transgenic male mice displayed osteopenia and impaired trabecular microarchitecture at 6 months of age. However, the inhibition of osteoblast number and activity in female mice did not result in changes in trabecular volume or structure. There is no immediate explanation for the discrepancy in the skeletal phenotype of male and female transgenics, and it is conceivable that mechanisms necessary for the protection of bone mass are at play in female mice in the context of HEY2 overexpression. Alternatively, a variable penetrance of the Col3.6-Hey2 transgene may be responsible for the absence of changes in trabecular bone of female transgenics.
The differences between the skeletal phenotypes observed in the two sexes also could be explained by a sexually dimorphic mechanism of HEY2 action in osteoblasts. HEY2 is a paralogue of HES1, and HES1 overexpression under the control of the 3.6-kb fragment of the Col1a1 promoter induces osteoclastogenesis in male mice and suppresses osteoblastogenesis in female mice, confirming that the skeletal effects of the HES and HEY families of proteins are sexually dimorphic (22). An explanation for the sexual dimorphism could be provided by the protective effects of estrogens on skeletal mass (53).
In vitro studies exploring the differentiation and function of osteoblastic cells from mice misexpressing Hey2 indicate that HEY2 is dispensable for osteoblastogenesis and that perturbation of its expression in mature cells suppresses osteoblast function in both male and female mice. Although the mechanism was not established, it is possible that HEY2, like HEY1, interacts with transcription factors necessary for osteoblastogenesis, such as Runx2 (15). The decreased osteoblastic function by Hey2 inactivation is not consistent with the effects of Notch on mature cells of the osteoblastic lineage. The suppressive effects of HEY2 on transcription might explain this discrepancy because the absence of HEY2 might lead to increased expression of inhibitors of osteoblastic function (1,3). Results from cells overexpressing HEY2 are in agreement with the inhibitory effects of Notch overexpression on osteoblast function (4). Overexpression of HEY2 in osteoblasts causes a less severe skeletal phenotype than the one observed following the induction of Notch under the control of the 3.6-kb fragment of the Col1a1 promoter. This may suggest that the skeletal effects of Notch require expression of additional targets of Notch signaling, such as HES1, or are primarily mediated by direct regulation of osteoblast-specific genes by Notch (16).
The increased bone resorption reported in Hey2 transgenic mice does not phenocopy the inhibitory effect of Notch on osteoclastogenesis, which is mediated by an induction of OPG and suppression of RANKL and M-CSF expression by osteoblastic cells (15,25,26,48). HES1 is an inducer of osteoclastogenesis, suggesting that HEY and HES transcription factors carry out functions that are independent from their role as targets of Notch signaling (22). We report an induction of Il6 expression by HEY2 in osteoblasts from male mice, suggesting a possible mechanism for the enhanced bone resorption observed in male Col3.6-Hey2 transgenics. However, HEY2, like Notch, induced OPG expression, and this may be a protective mechanism to prevent excessive osteoclastogenesis. IL6 serum levels were not increased in Col3.6-Hey2 transgenic mice (data not shown), indicating that IL6 acts locally to regulate bone resorption. Although IL6 regulates osteoclastogenesis by inducing RANKL in osteoblastic cells, its direct effects on cells of the osteoclast lineage are less clear, and both a stimulatory function and an inhibitory function of IL6 in the differentiation of osteoclast precursors have been reported (54,55). Our observations indicate that in the context of HEY2 overexpression, IL6 promotes the resorptive activity of osteoclast precursors.
In conclusion, Hey2 plays a modest role in the regulation of skeletal cell function, whereas HEY2 overexpression in male mice uncouples bone formation from bone resorption, causes osteopenia, and compromises bone microarchitecture.