The MAPK Erk5 is necessary for proper skeletogenesis involving a Smurf-Smad-Sox9 molecular axis.

Erk5 belongs to the mitogen-activated protein kinase (MAPK) family. Following its phosphorylation by Mek5, Erk5 modulates several signaling pathways in a number of cell types. In this study, we demonstrated that Erk5 inactivation in mesenchymal cells causes abnormalities in skeletal development by inducing Sox9, an important transcription factor of skeletogenesis. We further demonstrate that Erk5 directly phosphorylates and activates Smurf2 (a ubiquitin E3 ligase) at Thr249, which promotes the proteasomal degradation of Smad proteins and phosphorylates Smad1 at Ser206 in the linker region known to trigger its proteasomal degradation by Smurf1. Smads transcriptionally activated the expression of Sox9 in mesenchymal cells. Accordingly, removal of one Sox9 allele in mesenchymal cells from Erk5-deficient mice rescued some abnormalities of skeletogenesis. These findings highlight the importance of the Mek5-Erk5-Smurf-Smad-Sox9 axis in mammalian skeletogenesis.

Translational Control of Sox9 RNA by mTORC1 Contributes to Skeletogenesis.

The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) regulates cellular function in various cell types. Although the role of mTORC1 in skeletogenesis has been investigated previously, here we show a critical role of mTORC1/4E-BPs/SOX9 axis in regulating skeletogenesis through its expression in undifferentiated mesenchymal cells. Inactivation of Raptor, a component of mTORC1, in limb buds before mesenchymal condensations resulted in a marked loss of both cartilage and bone. Mechanistically, we demonstrated that mTORC1 selectively controls the RNA translation of Sox9, which harbors a 5' terminal oligopyrimidine tract motif, via inhibition of the 4E-BPs. Indeed, introduction of Sox9 or a knockdown of 4E-BP1/2 in undifferentiated mesenchymal cells markedly rescued the deficiency of the condensation observed in Raptor-deficient mice. Furthermore, introduction of the Sox9 transgene rescued phenotypes of deficient skeletal growth in Raptor-deficient mice. These findings highlight a critical role of mTORC1 in mammalian skeletogenesis, at least in part, through translational control of Sox9 RNA.

Hypoxic Stress Upregulates the Expression of Slc38a1 in Brown Adipocytes via Hypoxia-Inducible Factor-1α.

The availability of amino acid in the brown adipose tissue (BAT) has been shown to be altered under various conditions; however, little is known about the possible expression and pivotal role of amino acid transporters in BAT under physiological and pathological conditions. The present study comprehensively investigated whether amino acid transporters are regulated by obesogenic conditions in BAT in vivo. Moreover, we investigated the mechanism underlying the regulation of the expression of amino acid transporters by various stressors in brown adipocytes in vitro. The expression of solute carrier family 38 member 1 (Slc38a1; gene encoding sodium-coupled neutral amino acid transporter 1) was preferentially upregulated in the BAT of both genetic and acquired obesity mice in vivo. Moreover, the expression of Slc38a1 was induced by hypoxic stress through hypoxia-inducible factor-1α, which is a master transcription factor of the adaptive response to hypoxic stress, in brown adipocytes in vitro. These results indicate that Slc38a1 is an obesity-associated gene in BAT and a hypoxia-responsive gene in brown adipocytes.

Amelioration of the Development of Osteoarthritis by Daily Intake of ß-Cryptoxanthin.

ß-Cryptoxanthin, which is primarily obtained from citrus fruits such as Satsuma mandarins, is a major carotenoid routinely found in human serum. Recently, we demonstrated that daily oral intake of ß-cryptoxanthin prevented ovariectomy-induced bone loss and ameliorated neuropathic pain in mice. Although ß-cryptoxanthin exerts preventive effects on various lifestyle-related diseases, there have been no studies on the effect of ß-cryptoxanthin on the development of osteoarthritis, the most common degenerative joint disease, which frequently leads to loss of ability and stiffness in the elderly. Here we showed that daily oral administration of ß-cryptoxanthin significantly prevented the development of osteoarthritis developed by surgically inducing knee joint instability in mice in vivo. Furthermore, in vitro experiments revealed that ß-cryptoxanthin markedly inhibited the expression of inflammatory cytokines and enzymes critical for the degradation of the extracellular matrix in primary chondrocytes. Our results suggest that oral supplementation of ß-cryptoxanthin would be beneficial for the maintenance of joint health and as prophylaxis against osteoarthritis.

Daily oral intake of ß-cryptoxanthin ameliorates neuropathic pain.

ß-cryptoxanthin, a xanthophyll carotenoid, exerts preventive effects on various lifestyle-related diseases. Here, we found that daily oral administration of ß-cryptoxanthin significantly ameliorated the development of tactile allodynia following spinal nerve injury but was ineffective in mechanical allodynia in an inflammatory pain model in mice. Our results suggest that ß-cryptoxanthin supplementation would be beneficial for the prophylaxis of neuropathic pain.

The transcriptional modulator Ifrd1 controls PGC-1α expression under short-term adrenergic stimulation in brown adipocytes.

Sympathetic tone activates the function of classical brown adipocytes, which constitutively exist in the brown adipose tissue (BAT), and inducible brown adipocytes (so-called beige adipocytes), which sporadically reside within the white adipose tissue (WAT). Here we identified the transcriptional modulator interferon-related developmental regulator 1 (Ifrd1) as a negative regulator of thermogenic and mitochondrial gene expression in brown adipocytes. Ifrd1 expression was markedly induced by cold exposure and administration of CL-316243 (a ß3 adrenergic agonist) in interscapular brown adipose and inguinal subcutaneous WATs, but not in epididymal visceral WAT, in vivo. Adrenergic stimulation also induced Ifrd1 expression in brown adipocytes in a cAMP responsive element binding protein-dependent manner in vitro. CL-316243 injection markedly elevated thermogenic and mitochondrial gene expression, including peroxisome proliferator-activated receptor γ coactivator 1α (Pgc1a) in the subcutaneous WAT of Ifrd1 knockout mice compared with gene expression in wild-type mice. Pgc1a promoter activity enhanced by the transcription factor specificity protein 1 (Sp1) was markedly repressed by co-introduction of Ifrd1 in brown adipocytes, whereas the repression was markedly prevented by the addition of trichostatin A, a histone deacetylase inhibitor. Moreover, adrenergic stimulation induced complex formation between Ifrd1, Sp1 and mSIN3B, which is a component of the SIN complex containing histone deacetylase, in brown adipocytes. These findings, therefore, suggest that Ifrd1 could be a pivotal negative regulator of sympathetic regulation of thermogenic and mitochondrial gene expression in brown adipocytes by interacting with Sp1 and the mSIN3 complex.

The transcriptional modulator Ifrd1 is a negative regulator of BMP-2-dependent osteoblastogenesis.

We previously demonstrated that the transcriptional coactivator/repressor interferon-related developmental regulator 1 (Ifrd1) was expressed in osteoblasts and participated in the regulation of bone homeostasis. However, it remains unclear how Ifrd1 expression itself is regulated in osteoblasts. In the present study, we investigated the upstream regulatory mechanisms of Ifrd1 in osteoblasts during osteoblastogenesis. Ifrd1 protein expression and runt-related transcription factor 2, the master regulator of osteoblastogenesis, were markedly upregulated by bone morphogenetic protein 2 (BMP-2) stimulation in primary osteoblasts. Moreover, BMP-2 stimulation significantly induced Ifrd1 mRNA expression and promoter activity in osteoblasts. LDN193189, an inhibitor of activin-like kinase 2/3, almost completely inhibited the BMP-2-induced increase in Ifrd1 protein expression. There were at least two putative Smad-binding elements in the 5'-flanking region, which was highly conserved between mouse and human Ifrd1 genes. Co-introduction of both Smad4 and Smad1 significantly increased Ifrd1 promoter activity in osteoblasts. In addition, BMP-2 induced the recruitment of Smad1 to the Ifrd1 promoter in osteoblasts. Moreover, BMP-2-dependent osteoblastogenesis was further enhanced in Ifrd1 knocked-down osteoblasts, as determined by the intensity of Alizarin red stain and marker gene expression. These results suggest that BMP-2 directly induces Ifrd1 expression at the transcriptional level in osteoblasts via the Smad pathway, and Ifrd1 negatively regulates BMP-2-dependent osteoblastogenesis.

ATF3 controls proliferation of osteoclast precursor and bone remodeling.

Bone homeostasis is maintained by the sophisticated coupled actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Here we identify activating transcription factor 3 (ATF3) as a pivotal transcription factor for the regulation of bone resorption and bone remodeling under a pathological condition through modulating the proliferation of osteoclast precursors. The osteoclast precursor-specific deletion of ATF3 in mice led to the prevention of receptor activator of nuclear factor-κB (RANK) ligand (RANKL)-induced bone resorption and bone loss, although neither bone volume nor osteoclastic parameter were markedly altered in these knockout mice under the physiological condition. RANKL-dependent osteoclastogenesis was impaired in vitro in ATF3-deleted bone marrow macrophages (BMM). Mechanistically, the deficiency of ATF3 impaired the RANKL-induced transient increase in cell proliferation of osteoclast precursors in bone marrow in vivo as well as of BMM in vitro. Moreover, ATF3 regulated cyclin D1 mRNA expression though modulating activator protein-1-dependent transcription in the osteoclast precursor, and the introduction of cyclin D1 significantly rescued the impairment of osteoclastogenesis in ATF3-deleted BMM. Therefore, these findings suggest that ATF3 could have a pivotal role in osteoclastogenesis and bone homeostasis though modulating cell proliferation under pathological conditions, thereby providing a target for bone diseases.

Transcriptional Modulator Ifrd1 Regulates Osteoclast Differentiation through Enhancing the NF-κB/NFATc1 Pathway.

Bone homeostasis is maintained by the synergistic actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Here, we show that the transcriptional coactivator/repressor interferon-related developmental regulator 1 (Ifrd1) is expressed in osteoclast lineages and represents a component of the machinery that regulates bone homeostasis. Ifrd1 expression was transcriptionally regulated in preosteoclasts by receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) through activator protein 1. Global deletion of murine Ifrd1 increased bone formation and decreased bone resorption, leading to a higher bone mass. Deletion of Ifrd1 in osteoclast precursors prevented RANKL-induced bone loss, although no bone loss was observed under normal physiological conditions. RANKL-dependent osteoclastogenesis was impaired in vitro in Ifrd1-deleted bone marrow macrophages (BMMs). Ifrd1 deficiency increased the acetylation of p65 at residues K122 and K123 via the inhibition of histone deacetylase-dependent deacetylation in BMMs. This repressed the NF-κB-dependent transcription of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), an essential regulator of osteoclastogenesis. These findings suggest that an Ifrd1/NF-κB/NFATc1 axis plays a pivotal role in bone remodeling in vivo and represents a therapeutic target for bone diseases.