Increased Bone Mass in Smurf1-Deficient Mice
locus was disrupted through targeted homologous recombination (). This manipulation was expected to generate a genetic null allele, as it would give rise to a truncated protein product that lacks the two highly conserved WW domains and the HECT ubiquitin ligase domain (), which are indispensable for Smurf function (Zhu et al., 1999
; Lin et al., 2000
; Kavsak et al., 2000
; Zhang et al., 2001
). Homozygous mutant (Smurf1−/−
) mice were born at expected Mendelian ratio (see Table S1 in the Supplemental Data
available with this article online), had a normal life span, and were as fertile as their wild-type littermates. At the molecular level, however, the mutated Smurf1
transcript appeared to be less stable than its wild-type counterpart as it accumulated to a much lower level in total cellular RNA in contrast to a marked increase of the Smurf2
transcript (). Given the crucial inductive role by the signaling output of TGFβ/BMP, this compensatory increase of Smurf2 expression was likely to be causative to the seemingly normal embryonic development of Smurf1−/−
mice. Despite the lack of gross developmental abnormalities or health problems of the newborns, complete necropsy and histological examination of 20 Smurf1−/−
mice between ages 4 and 15 months revealed a thickening of diaphysis in more than 75% of long bones from the cohort (). No other significant alteration in skeletal morphology was observed (data not shown). Measurement of bone mineral density (BMD) showed that this thickening represented a bone mass increase primarily between the topographical sections 4 and 17 of the femurs (). Although newborn Smurf1−/−
mice began with a normally mineralized skeleton, the increase of BMD progressed with age, reaching 10%, 17%, and 20% more than that of control littermates by the ages of 4, 9, and 14 months, respectively. Little difference of BMD was seen in metaphysis or epiphysis at the either end of femurs (, sections 1–4 and 17–20), regions primarily consisting of trabecular bones. This bone mass increase is likely due to cell-autonomous causes rather than alteration in the production of bone-metabolizing hormones or a general endocrine disturbance, as no apparent change in the serum levels of parathyroid hormone, calcium, or phosphorus was observed (data not shown).
Increased Bone Mass in Smurf1−/− Mice
Enhanced Osteoblast Activity in Smurf1−/− Mice
To investigate the cellular basis of bone mass increase associated with loss of Smurf1 function, we performed detailed histomorphometric measurements in sections of undecalcified tibiae that were collected at the ages of 4 and 9 months. Similar to femurs, Smurf1−/− tibiae also exhibited a marked thickening of cortical bone. The ratio of cortex width to total bone diameter at the midpoint of tibiae, a two-dimensional approximation of cortical bone volume, was about 20% higher (, left panel). Likewise, the ratio of trabecular bone volume to tissue volume, a surrogate measure of trabecular bone mass, increased slightly at 4 months of age (13.13% versus 11.73%) but more dramatically at 9 months (4.54% versus 2.99%) (, right panel). The hitherto-described bone mass increase could not be attributed to the cartilage-dependent endochondral bone formation, as the length and the growth plate anatomic structure of Smurf1−/− long bones were indistinguishable from those of wild-type mice ( and and data not shown). In contrast, change was observed neither in the number of multinucleated osteoclasts that were stained positive for tartrate-resistant acid phosphatase (TRAP) (, left panel) nor in the number of osteoblasts in 1% Toluidine blue stained bone sections (, right panel). Thus, this ruled out an imbalance in osteoblast/osteoclast differentiation, which would have altered the ratio of the respective cell numbers as a likely cause of the observed bone mass increase. However, calcein double labeling analysis, a histomorphometrical measurement of osteoblast activity in vivo, revealed a significant increase in bone formation rate associated with loss of Smurf1 () while urinary elimination of deoxypyridinoline, a biochemical marker of osteoclast activity, was normal (data not shown). Taken together, these results indicate that the bone mass increase in Smurf1−/− mice likely emanates from an enhanced bone-forming activity of osteoblasts.
Enhanced Osteoblast Activity in Smurf1−/− Mice
To ascertain that the expression pattern of Smurf genes is compatible with a role in osteoblast, we conducted in situ hybridization analyses in tibiae sections. Signals of Smurf1 transcript were concentrated in osteoblasts and proliferative chondrocytes but not in osteoclasts (). Smurf2 transcript exhibited a similar cellular distribution, but its level in Smurf1−/− tibiae was markedly increased (). Real-time RT-PCR quantification of mRNA isolated from bone extracts or purified osteoblasts confirmed the increase of Smurf2 expression (). Apparently, the elevated Smurf2 was not sufficient to completely compensate for the loss of Smurf1 function that enhanced osteoblast activity.
Smurf1 Negatively Regulates Osteoblast Function and Response to BMP
A consequence of enhanced osteoblast activity would be acceleration of bone ECM production. Real-time RT-PCR analyses revealed that this indeed was the case. The mRNA levels of α1 collagen type 1, α2 collagen type 1, osteocalcin, and bone sialoprotein were all increased in bone extracts of Smurf1−/− mice (). In contrast, the mRNA level of Runx2, the early differentiation marker of commitment to osteoblastic lineage, was unchanged (). This observation was consistent with the normal number of osteoblasts described in above histomorphometric measurements ().
Enhanced ECM Production in Smurf1−/− Mice and BMP Sensitivity of Smurf1−/− Osteoblasts
To specifically address the impact of loss of Smurf1
on bone-forming activity of osteoblasts, we established an osteoblast culture using cells isolated from calvaria bones. In vitro differentiation of mesenchymal progenitor cells or immature osteoblasts that are enriched in this culture faithfully recapitulates the osteoblast maturation process in expressing alkaline phosphatases (ALP), depositing type I collagen to ECM, and forming mineralized bone nodules (Bhargava et al., 1988
). In Smurf1−/−
osteoblasts, although ALP activity was comparable to that of wild-type osteoblasts initially after culturing ex vivo for 7 days, it became significantly higher after 10 days (). Similarly, production of collagen matrix (van Gieson staining) was dramatically increased after 12 days (), and more and bigger mineralized bone nodules (von Kossa staining) appeared after 21 days (). Thus, disruption of Smurf1
clearly has an augmentative effect on osteoblast activity, indicating that Smurf1 normally is a negative regulator of osteoblast function.
Both TGFβ and BMP play important roles in osteoblast differentiation and function, and, as implicated by previous in vitro studies, Smurf1 has the potential to degrade the BMP pathway-specific Smads and BMP or TGFβ type I receptors (Zhu et al., 1999
; Ebisawa et al., 2001
; Murakami et al., 2003
; Ying et al., 2003
). We thus examined the effect of TGFβ or BMP on osteoblastic function in calvaria cell culture. At the seventh day of ex vivo culturing, little ALP activity was detected without ligand treatment (). In the presence of BMP-2, however, ALP activity was induced in wild-type cells at this early time point, but a much robust induction was observed in Smurf1−/−
cells (). In the presence of TGFβ, which inhibits osteoblast differentiation and function, the basal level of ALP activity was suppressed, and, when added together with BMP-2, TGFβ also suppressed the BMP-induced ALP activity by more than 80% in both types of cells (). These results indicate that Smurf1
-deficient osteoblasts were sensitized to BMP signaling while probably having a normal response to TGFβ.
Normal Smad-Dependent Responses in Smurf1-Deficient Osteoblasts
To further delineate the effect of loss of Smurf1
on TGFβ or BMP signaling in osteoblasts, we analyzed the signaling output of a number of pathway-specific transcriptional reporters that were transfected into calvaria cells. We began with (CAGA)12
-luc, which can be activated only by TGFβ through its type I receptor (Dennler et al., 1998
), and BRE-luc, a BMP-specific transcription reporter driven by BMP-responsive elements of the Id1
gene (Korchynskyi and ten Dijke, 2002
). While addition of either TGFβ or BMP-2 stimulated their respective reporters, the extent to which these reporters were activated was independent of the genetic background of the cells in which they were tested (). Western blot analyses showed little difference in the levels of total Smad2 and Smad3 (), Smad1 and Smad5, and the BMP type IA and IB receptors (). Although the BMP-2-mediated Smad1 and Smad5 phosphorylation lasted longer than that of the TGFβ-mediated Smad2 phosphorylation, no significant difference was observed between wild-type and Smurf1−/−
osteoblasts (). Thus, even though Smurf1
-deficient osteoblasts are sensitized to BMP for controlling bone-forming activity, the Smad-dependent TGFβ and BMP signaling per se is not affected. Notwithstanding, Smurf1 did display the ability to down-regulate the activities of these pathway-specific reporters when it was overexpressed (), in agreement with previous reports (Zhu et al., 1999
; Ebisawa et al., 2001
; Murakami et al., 2003
Normal Smad-Dependent TGFβ and BMP Response in Smurf1−/− Osteoblasts
Surprisingly, when the signaling response was measured with a set of more complex transcriptional reporters—OC-luc for monitoring BMP response and 3TP-lux for TGFβ response—a much potent transcriptional activation was now observed in Smurf1−/−
cells (). The control elements of OC-luc reporter were derived from the osteocalcin promoter (Ducy and Karsenty, 1995
), whose activation requires osteoblast-specific transcription factor Runx2 and is influenced by interaction between Runx2 and other coactivators, including Smads and AP-1 (Franceschi and Xiao, 2003
; Ito and Miyazono, 2003
). Without culturing in differentiation medium or introduction of exogenous Runx2, OC-luc remained silent (). In the presence of exogenous Runx2, however, OC-luc was activated by BMP in both wild-type and Smurf1−/−
cells, but the extent of activation was much higher in Smurf1−/−
cells (). Although it was reported that Smurf1 could cause degradation of Runx2 (Zhao et al., 2003
), and overexpression of Smurf1 did decrease the overall transcription activity from this reporter (), the augmented activation of OC-luc in Smurf1−/−
cells could not be accounted solely by such a mechanism because the protein level of Runx2 was unchanged (). Similarly, an enhancement of TGFβ-induced transcription activation was observed in Smurf1−/−
cells when assayed by 3TP-lux (), whose promoter contains three tandem repeats of the AP-1 binding element and a segment of the TGFβ-inducible PAI-1 promoter (Wrana et al., 1992
). Because the AP-1 family of transcription factors can cooperate with Runx2 or Smad to increase Runx2- or Smad-dependent transcription (Zhang et al., 1998
; Wong et al., 1999
; Hess et al., 2001
; D’Alonzo et al., 2002
), the above results suggest that Smurf1 may regulate osteoblast function and response to BMP by modulating AP-1 activity.
Activation of JNK Kinase Cascade in Smurf1-Deficient Osteoblasts
The overt increase of osteoblast activity in Smurf1−/−
mice and the seeming lack of change in the Smad-dependent signaling prompted us to look beyond the orthodox TGFβ/BMP intracellular signaling pathway for physiological target of Smurf1 function. The crosstalks between TGFβ/BMP signaling and various MAP kinases are well substantiated (Massagué, 2000
; Derynck and Zhang 2003
). Two MAP kinases, JNK and p38 MAPK, which act upstream of the AP-1 family transcription factors, have recently been shown to play a role in controlling bone ECM production, expression of osteoblast specific markers, and other aspects of osteoblast function (Guicheux et al., 2003
). Indeed, Western blot analyses showed that, while JNK could become phosphorylated in response to BMP in wild-type osteoblasts, it was already phosphorylated without the ligand in Smurf1−/−
cells (). The level of total JNK, however, remained constant (), suggesting that the appearance of phosphorylated JNK was due to post-translational activation. In contrast, little change was seen in the levels of either total or phosphorylated p38 MAPK (). Consequential to JNK activation, downstream JNK-dependent transcription was enhanced in Smurf1−/−
cells, as indicated by AP1-luc reporter assay (). In addition, the protein level of one of JNK targets, JunB, but not c-Jun or JunD was increased in Smurf1−/−
osteoblasts (), although neither c-Jun nor JunB itself constitutes as direct target for Smurf1 (Figure S1
). Functional cooperation between Smads and other families of transcription activators, including members of the AP-1 or ATF family, is a recurring theme in activation of TGFβ/BMP target genes (Massagué, 2000
; Derynck and Zhang 2003
). Thus, the activation of JNK and its downstream AP-1 or ATF effectors may account for the enhanced bone-forming activity as well as the increased sensitivity to BMP signaling in Smurf1−/−
osteoblasts. To test this hypothesis, we applied SP600125, a JNK-specific inhibitor, to the calvaria cell culture. Blocking JNK by SP600125 suppressed the accelerated bone ECM production and reduced the augmented ALP activity in Smurf1−/−
osteoblasts to a level that was comparable to that in wild-type cells (). This was specific to the inhibition of JNK because treating calvaria cells with SB203580, a p38 MAPK inhibitor, or Y27632, a Rho-dependent kinase inhibitor, had little effect (). Blocking JNK also specifically desensitized the osteoblasts to BMP signaling, as the amplitude of BMP-induced ALP activity was curtailed by the addition of SP600125 (). Based on the results above, we conclude that activation of the JNK kinase cascade is an essential molecular change that leads to enhanced bone-forming activity and sensitizes BMP response associated with the loss of Smurf1 function. Interestingly, although blocking p38 MAPK by SB203580 had little effect on basal level of bone ECM production and ALP activity (), it nevertheless dampened the BMP-induced ALP activity (), suggesting that p38 MAPK may play a role in the BMP-stimulated osteoblast activity.
Elevated JNK Activity in Smurf1−/−Osteoblasts
Smurf1 Physically Interacts with and Targets MEKK2 for Ubiquitination
Having established a requirement for the JNK kinase cascade, we set out to identify a direct target of Smurf1 that controls osteoblast activity and BMP response. Smurfs are known to recognize a “PY” motif that has the ability to interact with WW domains of the HECT family E3 ligases (Sudol and Hunter, 2000
). No such motif was present in JNK, nor did JNK interact physically with Smurf1 (data not shown). These preclude JNK itself as a direct Smurf1 target. We then scanned the sequence of several kinases that are known to act upstream of JNK, including MKK3, -4, -6, and -7; MEKK1, -2, -3, and -4; and TAK1 (Davis, 2000
). Among these, only MEKK2 and MEKK3 contain a PY motif. Western blot showed that a slow-migrating band of MEKK2 accumulated in Smurf1−/−
but not wild-type osteoblasts (). This slow-migrating band is likely to be the phosphorylated MEKK2, because introduction of the wild-type but not a kinase-deficient MEKK2 cDNA (Su et al., 2001
) into mouse embryonic fibroblasts (MEFs) gave rise to a similar slow-migrating band, which could be collapsed to the normal migrating band by λ-phosphatase treatment (). In contrast to the accumulation of phosphorylated MEKK2, no difference in the level of the related MEKK3 was observed (). No discernible change was seen in two other kinases, MEKK1 and TAK1, which were implicated previously in TGFβ/BMP signaling (Brown et al., 1999
; Yamaguchi et al., 1999
) (). Nor was the level of RhoA (), a small GTPase that was recently reported as a target of Smurf1 (Wang et al., 2003
) and could function upstream of JNK.
Accumulation of Phosphorylated MEKK2 in Smurf1−/− Osteoblasts and Physical Interaction between Smurf1 and MEKK2
To investigate if MEKK2 accumulates in the absence of Smurf1 due to impairment of protein turnover, we measured the turnover rate of exogenous MEKK2 in MEFs. Western analyses showed that the rate of MEKK2 turnover was indeed impeded in Smurf1−/−MEFs (). This was especially true to the phosphorylated MEKK2; however, the turnover rate of kinase-deficient MEKK2(KM) was unaffected (). Similar results were obtained with pulse-chase labeling experiments. In wild-type MEFs, MEKK2 displayed a half-life of less than 1 hr, which was prolonged to about 2 hr in Smurf1−/− MEFs, similar to that of MEKK2(KM) (). Taken together, the above results suggest that MEKK2 is a target of Smurf1.
To determine if Smurf1 specifically interacts with MEKK2, we coexpressed MEKK2 with Smurf1, Smurf2, or dominant-negative mutants carrying an inactivating point mutation in their respective HECT E3 ligase domain in Smurf1−/− MEFs. In agreement with the protein turnover studies, MEKK2 and its phosphorylated form ceased to accumulate when it was coexpressed with the wild-type Smurf1 (, lane 2) but accumulated to high level with Smurf1(DN) (lane 3). Once again, accumulation of the kinase-deficient MEKK2(KM) was unaffected by Smurf1 or Smurf1(DN) (, lanes 6–8). Coexpression of Smurf2 or Smurf2(DN) had little effect on MEKK2 (, lanes 4 and 5), indicating that turnover of MEKK2 was specifically controlled by Smurf1. Immunoprecipitation experiments showed that Smurf1(DN) interacts strongly with the HA-tagged MEKK2 (, lane 3) but weakly with MEKK2(KM) (lane 7). Consistent with the resistance to Smurf2, MEKK2 did not coprecipitate with Smurf2 (, lanes 4 and 5). We did not detect wild-type Smurf1 in the anti-HA-MEKK2 pellet (, lane 2), possibly because MEKK2 was rapidly degraded once it was recruited to Smurf1. To demonstrate the in vivo interaction between these two proteins, we pretreated calvaria cells with the proteasome inhibitors MG132 and MG115. A slow-migrating phosphorylated MEKK2 accumulated under this condition in the otherwise wild-type osteoblasts, and the endogenous Smurf1 was co-precipitated with the accumulated MEKK2 (). The hitherto described interaction appears to be of direct nature, as it is corroborated in yeast two-hybrid assay () in which no homolog of TGFβ/BMP receptors or Smads exists. Mapping studies showed that MEKK2 specifically interacts with the two WW domains of Smurf1 between residues 146 and 351 and that this binding requires the PY motif of MEKK2 ().
To formally demonstrate that MEKK2 is a substrate of Smurf1, we performed a series of ubiquitination assays. First, in wild-type calvaria cells, polyubiquitinated MEKK2 accumulated to a higher level than in Smurf1−/− cells in the presence of proteasome inhibitors (). Second, in transfected Smurf1−/− MEFs, we detected polyubiquitinated MEKK2 only when it was coexpressed with Smurf1 (). Accumulation of polyubiquitinated MEKK2 was dependent on its intrinsic kinase activity and the functionality of Smurf1 HECT E3 ligase (). Third, we reconstituted the ubiquitination of MEKK2 in vitro using MEKK2 isolated from transfected Hep3B cells, purified E1 and E2, and in vitro-translated Smurf1. In this reconstituted system, only the wild-type MEKK2 became polyubiquitinated when it was mixed with Smurf1 but not a truncated Smurf1 lacking the HECT domain (, lanes 2 and 1). The kinase-deficient MEKK2(KM) was refractory to Smurf1-mediated ubiquitination (, lanes 5 and 6). Pretreating the isolated MEKK2 with λ-phosphatase abolished its ubiquitination (, lanes 3 and 4), suggesting that Smurf1-mediated ubiquitination of MEKK2 requires phosphorylation. Similar results were obtained using purified recombinant GST-Smurf1 fusion protein from E. coli and FLAG-MEKK2 from Drosophila S2 cells ().
MEKK2 Is a Substrate of Smurf1-Mediated Ubiquitination and Activation of MEKK2-JNK Pathway Is Sufficient to Enhance Osteoblast Activity
Finally, we expressed constitutively active MEKK2 or JNK, or kinase-deficient MEKK2 in osteoblasts by retrovirus-mediated transduction (), and examined the effect of the ectopic expression on osteoblast activity. MEKK2(CT), the constitutively active form containing only the kinase domain (Cheng et al., 2000
), strongly activated JNK, as revealed by the ability of JNK1 from the infected calvaria cells to phosphorylate GST-c-Jun in an in vitro kinase assay (). MEKK2(CT) also induced high levels of ALP and collagen matrix production in both wild-type and Smurf1−/−
osteoblasts after they were put in differentiation medium (). Similarly, high levels of ALP activity and collagen matrix production were induced by JNKK2-JNK1, the constitutively active fusion protein (Zheng et al., 1999
) (). However, the kinase-deficient MEKK2(KM) reduced the production of ALP and collagen matrix in Smurf1−/−
cells while having little effect in wild-type osteoblasts (). We therefore conclude that MEKK2 is a bona fide substrate of Smurf1 and activation of the MEKK2-JNK signaling cascade in Smurf1−/−
mice is sufficient to enhance osteoblast activity.