Menin suppresses osteoblast differentiation by antagonizing the AP-1 factor, JunD.

Mice null for menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, exhibit cranial and facial hypoplasia suggesting a role for menin in bone formation. We have shown previously that menin is required for the commitment of multipotential mesenchymal stem cells into the osteoblast lineage in part by interacting with the bone morphogenetic protein (BMP)-2 signaling molecules Smad1/5, and the key osteoblast transcriptional regulator, Runx2 (Sowa H., Kaji, H., Hendy, G. N., Canaff, L., Komori, T., Sugimoto, T., and Chihara, K. (2004) J. Biol. Chem. 279, 40267-40275). However, menin inhibits the later differentiation of committed osteoblasts. The activator protein-1 (AP-1) transcription factor, JunD, is expressed in osteoblasts and has been shown to interact with menin in other cell types. Here, we examined the consequences of menin-JunD interaction on osteoblast differentiation in mouse osteoblastic MC3T3-E1 cells. JunD expression, assessed by immunoblot, gradually increased during osteoblast differentiation. Stable expression of JunD enhanced expression of the differentiation markers, Runx2, type 1 collagen (COL1), and osteocalcin (OCN) and alkaline phosphatase (ALP) activity and mineralization. Hence, JunD promotes osteoblast differentiation. In MC3T3-E1 cells in which menin expression was reduced by stable menin antisense DNA transfection, JunD levels were increased. When JunD and menin were co-transfected in MC3T3-E1 cells, they co-immunoprecipitated. JunD overexpression increased the transcriptional activity of an AP-1 luciferase reporter construct, and this activity was reduced by co-transfection of menin. Therefore, JunD and menin interact both physically and functionally in osteoblasts. Furthermore, menin overexpression inhibited the ALP activity induced by JunD. In conclusion, the data suggest that menin suppresses osteoblast maturation, in part, by inhibiting the differentiation actions of JunD.

The activator protein-1 (AP-1) 1 transcription factor complex plays an important role in skeletal development and maintenance (1). AP-1 consists of dimers formed by members of the Fos, Jun, and ATF protein families. Fos proteins (c-Fos, FosB, Fra-1, Fra-2) are only able to heterodimerize with members of the Jun family, whereas the Jun proteins (c-Jun, JunB, JunD) can both homo-and heterodimerize with Fos proteins to form transcriptionally active complexes. AP-1 is implicated in cell differentiation, proliferation, apoptosis, and oncogenic transformation (2). AP-1 activity is controlled by upstream kinases and is modulated by other transcription factors. The study of genetically modified mice (and cells) has provided some insights into the biological functions of AP-1 family members (1,3). Deletion of Fra1, c-Jun, or JunB in mice is embryonic lethal, whereas lack of c-Fos, FosB, or JunD is not. Thus, some Fos/ Jun proteins are essential and these and the others may have redundant or overlapping functions.
Overexpression of c-Fos in transgenic and chimeric mice specifically affects bone, cartilage, and hematopoietic cell development, and the mice develop osteosarcomas (4). Homozygous c-Fos knock-out mice are growth-retarded, lack osteoclasts, and develop osteopetrosis with deficient bone remodeling (5). Thus, c-Fos is an essential regulator of macrophage/osteoclast lineage determination and bone remodeling (6). Overexpression of either Fra-1 or ⌬FosB increases bone formation causing osteosclerosis in transgenic mice (7,8). ⌬FosB, a naturally occurring alternatively spliced product lacking the COOH-terminal part of FosB and hence a known transcriptional domain, nonetheless does influence transcriptional activity possibly by heterodimerizing with other AP-1 proteins such as JunD (8). Studies of engineered mice with altered Fra-2 expression indicates that this AP-1 factor plays important roles in both osteoblast and osteoclast differentiation. Bone volume is markedly reduced and both number and sizes of osteoclasts are dramatically increased in Fra-2 knockout mice (9). Loss of JunB in mice results in reduced bone formation and severe bone turnover osteopenia mainly due to a cell-autonomous osteoblast and osteoclast differentiation defect (10). The role that JunD might play in bone biology is less clear. It is known that while the various AP-1 family members are differentially expressed in osteoblasts in vitro, Fra-2 and JunD predominate in differentiating osteoblasts (11). Homozygous JunD Ϫ/Ϫ mice show reduced postnatal growth, although the nature of any altered bone phenotype has not yet been examined (12), and JunD Ϫ/Ϫ males exhibit defects in reproduction although no defects in fertility have been observed in JunD Ϫ/Ϫ females. Therefore, the evidence overall points to redundant functions for members of the Fos/Jun family including JunD during development.
Menin is the product of the multiple endocrine neoplasia type 1 (MEN1) gene, which when inactivated is responsible for an autosomal dominant cancer syndrome characterized by tumors of the parathyroid, endocrine pancreas, and anterior pituitary (13). The 610-amino acid protein has carboxyl-terminal nuclear localization sequences and has been demonstrated to be predominantly nuclear (14,15). The physiological functions of menin are unclear but may be related to transcriptional regulation (16,17), cell cycle control (15), and interactions with a variety of proteins including transcription factors have been demonstrated (16 -20). Menin is widely expressed from an early developmental stage and found in both nonendocrine and endocrine tissues (21).
Recently, Crabtree et al. (22) reported that whereas mice heterozygous for menin inactivation exhibit a phenotype similar to that of the human MEN1 disorder, and develop endocrine tumors later in life, homozygous menin inactivation was embryonic lethal, and some fetuses had clear defects in cranial and facial development. This suggested that menin might play a role in osteoblast formation and differentiation. We showed previously that menin is required for the commitment of multipotential mesenchymal stem cells to the osteoblast lineage (23). This occurred, in part, by the roles played by menin in facilitating BMP signaling via Smads and the transcriptional activity of the key osteoblast regulator, Runx2 (23,24). Thus, menin physically and functionally interacted with Smads1/5 and Runx2 in mesenchymal stem cells. In committed osteoblasts these interactions were, for the most part, lost and menin inhibited later osteoblastic differentiation. This seemed to occur, in part, by the interaction of menin with the TGF-␤/ Smad3 pathway (24). However, it is likely that other mechanisms are involved in the role of menin in retarding differentiation of mature osteoblasts.
JunD has been identified as an interacting partner of menin, and this interaction leads to a repression of JunD-activated transcription (16,25). Among several AP-1 transcription factors tested, only JunD directly binds to menin. The physiological significance of menin-JunD interaction, in any tissue, is not known. In the present study, we have examined the role of JunD in osteoblast differentiation by use of mouse MC3T3-E1 cells. Moreover, we demonstrated that JunD and menin interact in these osteoblast cells and explored the consequences of this interaction with respect to the suppressive effect of menin on the further differentiation of committed osteoblasts.

EXPERIMENTAL PROCEDURES
Materials-Human recombinant TGF-␤ was from Sigma. Anti-JunD antibody was from Sigma and Type I collagen antibody was from Calbiochem LSL Co., Ltd. (Tokyo, Japan). The menin rabbit polyclonal antibody was generated as described previously (15). All chemicals used were of analytical grade.
Construction of Expression Plasmids and Stable Transfection-The human sense menin cDNA expression vector was constructed as described previously (17). For the antisense menin construct, human menin DNA was cloned in an antisense orientation into the EcoRI site of pcDNA3.1(ϩ). An XbaI-HindIII fragment bearing the mouse JunD DNA was cloned into pcDNA3.1(Ϫ) to create the sense JunD cDNA construct. Mouse JunD DNA, antisense menin DNA (AS), or empty vector (V) (each 3 g) were transfected into MC3T3-E1 cells with Lipofectamine (Invitrogen). Six hours after transfection, the cells were fed with fresh ␣-minimal essential medium containing 10% FBS. After 48 h, cells were passaged, and clones were selected in ␣-minimal essential medium supplemented with G418 (0.3 mg/ml) (Invitrogen) and 10% FBS. Reduced expression of menin by AS-DNA was detected with immunoblot analysis, using the polyclonal anti-menin antibody. To rule out the possibility of clonal variation, we characterized at least three independent clones for each transfection. V-transfected cells were used as the control.
Luciferase Assay-Cells were seeded at a density of 2 ϫ 10 5 /6-well plate. Twenty-four hours later, cells were transfected with 3 g of the reporter plasmid (p3TP-Lux) and the pCH110 plasmid expressing ␤-galactosidase (1 g) using Lipofectamine. Fifteen hours later, the medium was changed to a 4% FBS-containing medium, and the cells were incubated for an additional 9 h. Thereafter, the cells were cultured for 24 h in the presence or absence of 5 ng/ml TGF-␤ in medium containing 0.2% FBS. Cells were lysed, and the luciferase activity measured and normalized to the relative ␣-galactosidase activity, as described (17).
Protein Extraction, Co-immunoprecipitation, and Western Blot Analysis-Cells were lysed with radioimmunoprecipitation buffer containing 0.5 mM phenylmethylsulfonyl fluoride, complete protease inhibitor mixture, 1% Triton X-100, and 1 mM sodium orthovanadate. Cell lysates were centrifuged at 12,000 ϫ g for 20 min at 4°C, and the supernatants were stored at Ϫ80°C. Protein quantitation was performed with BCA protein assay reagent (Pierce). Proteins were denatured in SDS sample buffer and separated on 10% polyacrylamide-SDS gels and then transferred in 25 mM Tris, 192 mM glycine, and 20% methanol to polyvinylidene difluoride. Blots were blocked with TBS (20 mM Tris-HCl (pH 7.5) and 137 mM NaCl) plus 0.1% Tween 20 containing 3% dried milk powder. The antigen-antibody complexes were visualized using the appropriate secondary antibodies (Sigma) and the enhanced chemiluminescence detection system, as recommended by the manufacturer (Amersham Biosciences). For all experiments, 20 g of protein were applied to each lane.
For co-immunoprecipitation experiments, cells were lysed with a buffer containing 1% Triton X-100, 1% deoxycholate, 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 25 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1.5 mM MgCl 2 , 2 mM EGTA, plus protease inhibitor mixture for 30 min at 4°C, and insoluble materials were separated by centrifugation at 4°C for 30 min at 14,000 ϫ g. An aliquot of the supernatant (1 mg of protein) was clarified and incubated with anti-JunD antibody on a rocking platform at 4°C overnight. The immune complexes were collected with protein G Plus/ protein A-agarose beads (Calbiochem) for 30 min at 4°C. The beads were washed three times with the lysis buffer, resuspended in 2ϫ sample buffer, and boiled for 5 min. Immunoprecipitated proteins were then analyzed by SDS-PAGE and subjected to Western blot analysis, as described above.
RNA Extraction and Northern Blot Analysis-RNA was prepared from cells with TRIzol reagent (Invitrogen). Northern blot analysis was performed, as described previously (26). In brief, 20-g aliquots of total RNA were denatured, electrophoresed on 1% agarose gels containing 2% formaldehyde, and then transferred to nitrocellulose membranes and fixed with UV light. Membranes were hybridized to a 32 P-labeled DNA probe overnight at 42°C. The hybridization probes were type I collagen (COL1) (a gift from Dr. T. Kimura, Osaka University, Osaka, Japan) and mouse OCN. After hybridization, the filters were washed twice with saline citrate containing SDS and exposed to x-ray film. All values were normalized for RNA loading by probing blots with human ␤-actin cDNA (Wako Pure Chemical Industries, Ltd., Osaka, Japan).
ALP Activity Assay and ALP Staining-ALP activity was assayed as described previously (27). In brief, the assay mixtures contained 0.1 M 2-amino-2-methyl-1-propanol (Sigma), 1 mM MgCl 2 , 8 mM p-nitrophenyl phosphate disodium, and cell homogenates. After a 3-min incubation at 37°C, the reaction was stopped with 0.1 N NaOH, and the absorbance was read at 405 nm. A standard curve was prepared with p-nitrophenol (Sigma). Each value was normalized to the protein concentration. ALP staining was performed by a standard protocol. In brief, cultured cells were rinsed in phosphate-buffered saline, fixed in 100% methanol, rinsed with phosphate-buffered saline, and then overlaid with 1.5 ml of 0.15 mg/ml 5-bromo-4-chloro-3-indolylphosphate (Invitrogen) plus 0.3 mg/ml nitro blue tetrazolium chloride (Invitrogen) in 0.1 M Tris-HCl (pH 9.5), 0.01 N NaOH, 0.05 M MgCl 2 , followed by incubation at room temperature for 2 h in the dark.
Mineralization Assay-Mineralization of MC3T3-E1 cells was determined in 6-and 12-well plates using von Kossa staining and Alizarin red staining, respectively. The cells were fixed with 95% ethanol and stained with AgNO 3 by the von Kossa method. At the same time, the 12-well plates were fixed with ice-cold 70% ethanol and stained with Alizarin red (Sigma). For quantitation, cells stained with Alizarin red were destained with ethylpyridinium chloride (Wako Pure Chemical Industries, Ltd.), and then the extracted stain was transferred to a 96-well plate, and the absorbance at 562 nm was measured using a microplate reader, as described previously (26).
Statistics-Data are expressed as means Ϯ S.E. Statistical analysis was performed using an unpaired t test or analysis of variance.

JunD Expression and Osteoblast
Differentiation-To examine the role of JunD in the later differentiation of osteoblastic cells, we employed MC3T3-E1 cells. We have previously shown that in MC3T3-E1 cells ALP activity increased with time of culture (up to 21 days). The levels of COL1 and OPN expression were highest at days 7 and 14 of culture, respectively, whereas the expression of OCN mRNA, a terminal differentiation marker, was highest at day 21 of culture (see Fig. 9, B and C, of Ref. 23). We confirmed these changes in the present study (data not shown) and demonstrated that endogenous JunD expression, detected by immunoblot, increased throughout the culture period to reach its highest level in the terminally differentiated 21-day culture (Fig. 1, A and B). These findings suggest that JunD plays an important role in well differentiated osteoblasts.
Generation of MC3T3-E1 Clones Stably Overexpressing JunD-To investigate the role of JunD in osteoblasts we first generated a set of MC3T3-E1 clones overexpressing JunD. Twenty-four clones of cells transfected with either JunD or empty vector were picked after 3 weeks of selection with G418. The clones were screened by immunoblot with anti-JunD antibody. Several clones of the JunD transfected cells showed elevated JunD expression relative to empty vector transfected cells ( Fig. 1C; clones #3, #9, and #12). These clones were used for further studies.
JunD Promotes Osteoblast Differentiation-To examine the effects of JunD overexpression on osteoblast differentiation we analyzed osteoblast-specific gene expression. The transcription factor, Runx2, is a master regulator of osteoblast differentiation. The expression of Runx2 was clearly increased in the JunD-stably transfected clones at day 7 of culture ( Fig. 2A). At this time, levels of both COL1 and OCN mRNA were higher in JunD-transfected clones than in empty vector clones (Fig. 2B). This was reflected in higher COL1 protein levels in JunD-transfected clones, compared with the control clones (Fig. 2C). ALP plays an important role in osteoblast function and, like COL1, is an osteoblast differentiation marker. ALP staining and activity were markedly increased in the JunD-overexpressing MC3T3-E1 clones relative to empty vector-transfected controls (Fig. 3, A and B). Taken together, these findings strongly suggest that JunD promotes osteoblast differentiation.
JunD Promotes Mineralization-Extracellular matrix mineralization is the most important phenomenon in bone formation. Accelerated mineralization occurred in the JunD-overexpressing MC3T3-E1 clones as assessed by both the von Kossa and Alizarin red methods (Fig. 4A). A quantitative analysis of the Alizarin red result is shown in Fig. 4B. The data indicate that JunD overexpression enhances osteoblast mineralization.
Menin-JunD Interaction and Osteoblast Differentiation-Menin is known to directly interact with JunD and inhibit its transcription although this has not been investigated in osteoblasts. We have shown that menin promotes the commitment of multipotential mesenchymal stem cells to the osteoblast lineage, but inhibits their later differentiation (23). In that study, menin inactivation achieved by menin antisense DNA stimulated osteoblast differentiation and mineralization in MC3T3-E1 cells. Therefore, the effects of JunD overexpression observed in the present study are analogous to those seen with menin inactivation in our previous study (23). We hypothesized

FIG. 1. Endogenous JunD expression in MC3T3-E1 cells. A
shows the endogenous expression of JunD in MC3T3-E1 cells. MC3T3-E1 cells were grown until the indicated times, total protein was extracted, and Western blot analysis was performed with anti-JunD antibody as described under "Experimental Procedures." B shows the quantitation of the analysis in A. The immunoblot signals were scanned and quantitated with NIH Image analyzer. Each JunD value was normalized to the relative level of ␤-actin. C shows the stable overexpression of JunD in MC3T3-E1 cells. After JunD-transfected (clones #3, #9, and #12) and vector-transfected MC3T3-E1 cells were cultured for 7 days, total protein was extracted, and Western blot analysis was performed as described under "Experimental Procedures." therefore that menin might suppress osteoblast differentiation by antagonizing the actions of JunD. To test our hypothesis, we first examined the effects of menin inactivation on endogenous JunD expression in MC3T3-E1 cells. Second, we examined the physical and functional interactions between JunD and menin.
We generated menin-inactivated MC3T3-E1 cell clones (AS-MC) by stable expression of menin antisense DNA (23). JunD expression, assessed by immunoblot, was higher in the menininactivated cells compared with empty vector-transfected cells (Fig. 5A). These results indicate that menin inactivation stimulates JunD expression in osteoblasts.
The promoter-reporter construct, 3TP-lux, is driven by increased AP-1 activity. The luciferase activity of the reporter was increased 4-fold in MC3T3-E1 cells in which the JunD construct had been transfected relative to the vector-alone control (Fig. 6A). However, co-transfection of a menin sense construct, while having no affect on basal luciferase activity, led to complete abrogation of the JunD-stimulated increase (Fig. 6A). Overall, these results suggest that JunD interacts both physically and functionally with menin in osteoblasts.
Finally, we investigated whether menin affects JunD-induced differentiation in osteoblasts. Co-transfection of menin significantly inhibited the ALP activity induced by JunD transfection in MC3T3-E1 cells (Fig. 6B). The data indicate that

FIG. 2. Effects of JunD on the expression of Runx2, bone matrix proteins, and ALP in MC3T3-E1 cells. A, effect of JunD on
Runx2 expression in MC3T3-E1 cells. After cells were grown for 7 days, total RNA was extracted, and semi-quantitative RT-PCR was performed as described under "Experimental Procedures." B, vector-transfected or JunD-transfected MC3T3-E1 cells were grown for 7 days, total RNA was extracted, and Northern blot analysis was performed for COL1, OCN, and ␣-actin mRNAs as described under "Experimental Procedures." C, vector-transfected (clones #1 and #2) and JunD-transfected (clones #3, #9, and #12) MC3T3-E1 cells were cultured for 7 days, total protein was extracted, and Western blot analysis was performed with anti-COL1 and ␤-actin antibodies as described under "Experimental Procedures."

FIG. 3. Effects of JunD on ALP in MC3T3-E1 cells.
Cells were grown for 7 days and ALP staining was performed (A) or ALP activity was measured (B) as described under "Experimental Procedures." Each value is the mean Ϯ S.E. of four determination. *, p Ͻ 0.01, compared with the vector-alone-transfected group.

FIG. 4. Effects of JunD on mineralization in MC3T3-E1 cells.
Confluent cells were cultured in medium with 10 mM ␣-glycerophosphate for 14 days. A, then cells were stained by the von Kossa method or with Alizarin red as described under "Experimental Procedures." B, cell layers stained with Alizarin red were destained, and the mineralization was quantitated as described under "Experimental Procedures." *, p Ͻ 0.01, compared with the vector-alone-transfected group. menin inhibits osteoblast differentiation by suppressing JunDinduced transcriptional activity and JunD expression. DISCUSSION In the present study, we show that JunD plays an important role in bone formation and stimulates differentiation and mineralization of osteblasts. JunD enhanced the expression of the key transcriptional regulator Runx2 and that of the differentiation markers, COL1 and OCN, and ALP activity and mineralization in mouse osteoblast MC3T3-E1 cells.
Unlike the majority of the Fos/Jun family members, JunD is antimitogenic. The expression of JunD is regulated in a cell cycle-dependent manner in some cells (5,15,28) and is generally constitutive being relatively refractory to growth factor or hormone stimulation (29). The full range of physiological roles played by JunD is not clear. Mice with disruption of the JunD gene develop post-weaning growth retardation and male mice have impaired spermatogenesis and reproductive defects (12). Recent studies indicate that JunD mediates survival signaling by the c-Jun NH 2 -terminal kinase (JNK) signal transduction pathway (30) and protects against chronic kidney disease by regulating paracrine mitogens (31). It has been proposed that JunD is part of a network controlling proliferation and preventing pathological progression in renal disease, for example (31). These recent findings suggest that JunD is involved in important physiological processes in several organs, although the loss of JunD may be compensated for by other factors in JunD knock-out mice (12).
With respect to bone, several JunD-related actions have been noted. The expression of JunD in osteoblasts can be modulated by external stimuli, either mechanical strain (32) or basic fibroblast growth factor (33). In the latter case, JunD is one of several AP-1 factors that may regulate collagenase-3 gene tran-scription in response to the growth factor (33). Overexpression of JunD and Fra2 represses Runx2-induced collagenase-3 gene promoter activity in differentiated osteoblasts suggesting that Runx2/AP-1 interaction regulates this matrix metalloprotease important for skeletal development and normal and pathological remodeling of bone (34). Moreover, expression of osteocalcin, the most abundant osteoblast-specific non-collagenous protein, is up-regulated by overexpression of JunD and Fra2 in rat osteoblasts (11). A recent study suggested that diminished AP-1 activity, especially JunD, and the resultant decline in the expression of the osteogenic cytokine, interleukin-11 (IL-11), by bone marrow stromal cells plays a role in the impaired bone formation of a strain of senescence-accelerated mice, an animal model of senile osteoporosis (35). However, there have been no reports as yet on the effects on JunD itself on osteoblast differentiation and mineralization.
The protein product of the multiple endocrine neoplasia type 1 gene, menin, is being intensively investigated with respect to identifying those properties responsible for its action as a tumor suppressor (18). However, it is also evident that menin has other physiological roles, for example, in modulating production of hormones such as parathyroid hormone (36), prolactin (37,38), and insulin (39). With respect to functions of menin in bone, we recently showed that menin inactivation specifically inhibits the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage (23). In the mesenchymal stem

FIG. 5. Effect of menin inactivation on expression of JunD and physical interaction of menin and JunD in MC3T3-E1 cells. A,
after cells were grown for 7 days, total protein was extracted, and Western blot analysis was performed as described under "Experimental Procedures." B, menin and/or JunD were transfected into MC3T3-E1 cells. Cell extracts were immunoprecipitated (IP) with anti-JunD antibody, followed by immunoblotting (IB) with anti-menin antibody, as described under "Experimental Procedures." Whole cell extracts were immunoblotted with anti-JunD and anti-menin antibodies.

FIG. 6. Functional interaction of menin and JunD in MC3T3-E1 cells.
A, the AP-1 responsive 3TP-lux promoter-reporter construct was transfected into MC3T3-E1 cells with empty vector, JunD, and/or menin expression plasmids, and relative luciferase activity was measured as described under "Experimental Procedures." Each value is the mean Ϯ S.E. of four determination. *, p Ͻ 0.01, compared with the vector alone-transfected group. B, effect of menin on JunDstimulated ALP activity in MC3T3-E1 cells. Menin was transfected into empty vector or JunD stably transfected MC3T3-E1 cells, and after 48 h, ALP activity was measured as described under "Experimental Procedures." Each value is the mean Ϯ S.E. of four determination. *, p Ͻ 0.01, compared with corresponding empty vector stably transfected group. **, p Ͻ 0.01, compared with the corresponding non-menintransfected group. cells, not yet committed to the osteoblast lineage, menin interacts with the crucial mediators of BMP-2 signaling, Smad1 and Smad5, as well as the key osteoblast transcriptional regulator, Runx2, to promote osteoblast commitment (23,24). In committed osteoblasts, these functional interactions are lost and menin inhibits the later osteoblast differentiation. Although this occurs, in part, by the interaction of menin with the TGF-␤/ Smad3 pathway (24), it appears likely that other mechanisms are also involved in the ability of menin to retard the differentiation of committed osteoblasts. This we have investigated in the present study.
Previous studies in nonosteoblast cells showed that menin interacts directly with JunD and inhibits JunD-activated gene transcription (16,25). Only the full-length isoform of JunD binds to menin and is considered to be its functional target (40). Menin represses JunD-mediated transcriptional activity by association with an mSin3A-histone deacetylase complex (41). The inhibition of the activity of one anti-mitogenic protein (JunD) by another, namely menin, appears paradoxical (15). However, it has recently been suggested that the presence of menin is important for the growth suppressor activity of JunD and in the absence of menin JunD switches from a growth suppressor to a growth promoter (42). However, the broader significance of menin/JunD interaction unrelated to tumor suppressor function is not known. We are demonstrating in our study, for the first time, the importance of menin/JunD interaction to osteoblast biology. Even though a particular proteinprotein interaction has been shown for one type of cell, this may not hold as one moves to other cell types. Therefore, it is important to explore the mechanism for each cell type examined.
In MC3T3-E1 cells as osteoblast differentiation progresses menin is highly expressed at days 7 and 14 days of culture but is decreased at day 21 (23). As demonstrated in the present study, the inverse pattern is seen with respect to JunD with a modest level of expression occurring at day 7 but then increasing through day 14 to day 21. To examine the role of JunD in osteoblasts we generated clones of MC3T3-E1 cells stably overexpressing JunD. These cells demonstrated enhanced expression of the differentiation markers, Runx2, COL1, and OCN and ALP activity and mineralization. This emphasized the important role played by JunD in promoting osteoblast differentiation. The observed phenotype was strikingly similar to that seen in our previous study in which inactivation of menin in MC3T3-E1 cells, achieved by stable transfection of antisense menin cDNA, enhanced differentiation marker expression and ALP activity and mineralization (23). This raised the hypothesis that menin might suppress osteoblast maturation by antagonizing the differentiating actions of JunD. As noted above, there is a normal reciprocal relationship between the expression levels of menin and JunD with the latter predominating in the most differentiated osteoblasts. This reciprocity of expression was reinforced in the present study with the demonstration that MC3T3-E1 clones stably expressing antisense menin cDNA had increased levels of JunD.
We then demonstrated that menin and JunD physically and functionally interact in osteoblasts. Menin suppressed JunDinduced transcriptional activity and inhibited ALP activity in the MC3T3-E1 cells. Therefore, menin suppresses osteoblast maturation, in part, by inhibiting the bone anabolic actions of JunD. This is the first evidence of menin-JunD interaction having important consequences distinct from anti-tumor effects. Interaction of menin and JunD may have a similar role in tissues other than bone.
Given our previous findings linking menin and the actions of TGF-␤ superfamily members to the commitment and differen-tiation of osteoblasts (23,24), the known involvement of JunD in TGF-␤ action in cells (other than osteoblasts) (43)(44)(45)(46)(47), and the present demonstration of critical menin-JunD interactions in osteoblasts it will be important to study these mechanisms further. The potential cross-talk between these pathways is currently being examined in our laboratories.