Yoshifumi Takahata

Functional Expression of β2 Adrenergic Receptors Responsible for Protection Against Oxidative Stress Through Promotion of Glutathione Synthesis After Nrf2 Upregulation in Undifferentiated Mesenchymal C3H10T1/2 Stem Cells

Adrenaline is believed to play a dual role as a neurotransmitter in the central nervous system and an adrenomedullary hormone in the peripheral tissues. In contrast to accumulating evidence for the involvement in endochondral ossification, osteoblastogenesis, and osteoclastogenesis, little attention has been paid to the role of adrenergic signals in the mechanisms underlying proliferation and differentiation of mesenchymal stem cells with self‐renewal capacity and multi‐potentiality to differentiate into osteoblast, chondrocyte, adipocyte, and myocyte lineages. Expression of mRNA was seen for different adrenergic receptor (AdR) subtypes, including β2AdR, in the mesenchymal stem cell line C3H10T1/2 cells and mouse bone marrow mesenchymal stem cells before differentiation. Exposure to adrenaline not only increased cAMP formation, phosphorylation of cAMP responsive element (CRE) binding protein (CREB) on serine133 and CRE reporter activity in a manner sensitive to propranolol, but also rendered C3H10T1/2 cells resistant to the cytotoxicity of hydrogen peroxide, but not of either 2,4‐dinitirophenol or tunicamycin. Adrenaline induced a rapid but transient increase in mRNA expression of the antioxidative gene nuclear factor E2 p45‐related factor‐2 (Nrf2) along with an increase in the cystine/glutamate antiporter subunit xCT mRNA expression. Hydrogen peroxide was less cytotoxic in cells overexpressing Nrf2, moreover, while adrenaline significantly increased xCT promoter activity with an increase in endogenous glutathione levels. These results suggest that adrenaline may selectively protect mesenchymal C3H10T1/2 cells from oxidative stress through a mechanism related to the promoted biosynthesis of glutathione in association with transient Nrf2 expression after activation of β2AdR. J. Cell. Physiol. 218: 268–275, 2009.

Modulation of cellular proliferation and differentiation through GABAB receptors expressed by undifferentiated neural progenitor cells isolated from fetal mouse brain

In this study, we have attempted to evaluate the possible role of metabotropic GABAB receptors (GABABR) expressed by neural progenitor cells prepared from neocortex of embryonic Std‐ddY mice. Immunocytochemical analysis confirmed the validity of isolation procedures of neural progenitors, while round spheres were formed with clustered cells during culture with epidermal growth factor (EGF) for 10 days. A reverse transcription polymerase chain reaction analysis revealed constitutive expression of GABAAR, GABABR, and GABACR subtypes in undifferentiated progenitors and neurospheres formed within 10 days. Exposure to GABA led to concentration‐dependent increases in the total area and proliferation activity of neurospheres at 10–300 µM, while the GABABR agonist baclofen at 100 µM significantly increased the size of neurospheres expressing both GABABR1 and GABABR2 subunits in a manner sensitive to a GABABR antagonist. By contrast, a significant decrease was seen in the total areas of neurospheres prepared from mice deficient of the GABABR1 subunit. In neurospheres of GABABR1‐null mice, a significant increase was induced in the number of cells immunoreactive for a glial marker protein, with a concomitant decrease in that of a neuronal marker protein, upon spontaneous differentiation after the removal of EGF. These results suggest that GABABR may be functionally expressed by neural progenitor cells to preferentially promote the commitment toward a neuronal lineage after the activation of cellular proliferation toward self‐replication in the developing mouse brain. J. Cell. Physiol. 216: 507–519, 2008.

Positive regulation of osteoclastic differentiation by growth differentiation factor 15 upregulated in osteocytic cells under hypoxia

Osteocytes are thought to play a role as a mechanical sensor through their communication network in bone. Although osteocytes are the most abundant cells in bone, little attention has been paid to their physiological and pathological functions in skeletogenesis. Here, we have attempted to delineate the pivotal functional role of osteocytes in regulation of bone remodeling under pathological conditions. We first found markedly increased osteoclastic differentiation by conditioned media (CM) from osteocytic MLO‐Y4 cells previously exposed to hypoxia in vitro. Using microarray and real‐time PCR analyses, we identified growth differentiation factor 15 (GDF15) as a key candidate factor secreted from osteocytes under hypoxia. Recombinant GDF15 significantly promoted osteoclastic differentiation in a concentration‐dependent manner, with concomitant facilitation of phosphorylation of both p65 and inhibitory‐κB in the presence of receptor activator of nuclear factor‐κB ligand. To examine the possible functional significance of GDF15 in vivo, mice were subjected to ligation of the right femoral artery as a hypoxic model. A significant increase in GDF15 expression was specifically observed in tibias of the ligated limb but not in tibias of the normally perfused limb. Under these experimental conditions, in cancellous bone of proximal tibias in the ligated limb, a significant reduction was observed in bone volume, whereas a significant increase was seen in the extent of osteoclast surface/bone surface when determined by bone histomorphometric analysis. Finally, the anti‐GDF15 antibody prevented bone loss through inhibiting osteoclastic activation in tibias from mice with femoral artery ligation in vivo, in addition to suppressing osteoclastic activity enhanced by CM from osteocytes exposed to hypoxia in vitro. These findings suggest that GDF15 could play a pivotal role in the pathogenesis of bone loss relevant to hypoxia through promotion of osteoclastogenesis after secretion from adjacent osteocytes during disuse and/or ischemia in bone.

The natural polyamines spermidine and spermine prevent bone loss through preferential disruption of osteoclastic activation in ovariectomized mice

Although naturally occurring polyamines are indispensable for a variety of cellular events in eukaryotic cells, little attention has been paid to their physiological and pathological significance in bone remodelling to date. In this study, we evaluated the pharmacological properties of several natural polyamines on the functionality and integrity of bone in both in vitro and in vivo experiments.

Repression of adipogenesis through promotion of Wnt/β-catenin signaling by TIS7 up-regulated in adipocytes under hypoxia

Although tetradecanoyl phorbol acetate induced sequence-7 (TIS7) has been identified as a co-activator/repressor of gene transcription in different eukaryotic cells, little attention has been paid to the functionality of TIS7 in adipocytes. Here, we evaluated the possible role of TIS7 in mechanisms underlying the regulation of adipogenesis. TIS7 expression was preferentially up-regulated in white adipose tissues (WAT) of obesity model mice as well as in pre-adipocytic 3T3-L1 cells cultured under hypoxic conditions. TIS7 promoter activity was selectively enhanced by activating transcription factor-6 (ATF6) among different transcription factors tested, while induction of TIS7 by hypoxic stress was markedly prevented by knockdown of ATF6 by shRNA in 3T3-L1 cells. Overexpression of TIS7 markedly inhibited Oil Red O staining and expression of particular adipogenic genes in 3T3-L1 cells. TIS7 synergistically promoted gene transactivation mediated by Wingless-type mouse mammary tumor virus integration site family (Wnt)/β-catenin, while blockade of the Wnt/β-catenin pathway by a dominant negative form of T-cell factor-4 (DN-TCF4) markedly prevented the inhibition of adipogenesis in 3T3-L1 cells with TIS7 overexpression. TIS7 predominantly interacted with β-catenin in the nucleus of WAT in the genetically obese ob/ob mice as well as in 3T3-L1 cells cultured under hypoxic conditions. Both knockdown of TIS7 by shRNA and introduction of DN-TCF4 similarly reversed the hypoxia-induced inhibition of adipogenic gene expression in 3T3-L1 cells. These findings suggest that TIS7 could play a pivotal role in adipogenesis through interacting with β-catenin to promote the canonical Wnt signaling in pre-adipocytes under hypoxic stress such as obesity.

Interference with cellular differentiation by D-serine through antagonism at N-methyl-D-aspartate receptors composed of NR1 and NR3A subunits in chondrocytes.

Serine racemase (SR) is responsible for the biosynthesis of D‐serine (D‐Ser), an endogenous co‐agonist for the glycine (Gly)‐binding site on N‐methyl‐D‐aspartate (NMDA) receptors, from L‐Ser in the brain. We have previously demonstrated high expression of SR by chondrocytes in cartilage. In this study, we attempted to elucidate the possible functional role of D‐Ser in chondrogenesis. Expression of mRNA and corresponding protein was seen for SR in cultured rat costal chondrocytes, while the addition of L‐Ser significantly increased intracellular and extracellular levels of D‐Ser. In organotypic cultured mouse embryonic metatarsals isolated before vascularization, SR mRNA was highly localized in hypertrophic and calcified chondrocytes. Exposure to D‐Ser not only suppressed several chondrocytic maturation markers, including alkaline phosphatase (ALP) activity, Ca2+ accumulation, nodule formation, and osteopontin expression, in rat chondrocytes, but also delayed chondral mineralization in mouse metatarsals. Either NMDA or Gly alone significantly increased Ca2+ accumulation in cultured chondrocytes, whereas D‐Ser significantly prevented Ca2+ accumulation by Gly, but not by NMDA. Gly alone also significantly increased gene transactivation by the introduction of runt‐related transcription factor‐2 (Runx2) in COS7 cells transfected with NR1 and NR3A subunits, while D‐Ser significantly prevented the increase by Gly without affecting the promoter activity of Runx2. In both cultured chondrocytes and metatarsals from NR1‐null mice, significant decreases were seen in ALP activity and chondral mineralization, respectively. These results suggest that D‐Ser may negatively regulate cellular differentiation through inhibiting NMDA receptors composed of NR1 and NR3A subunits in a manner related to Runx2 transcriptional activity in chondrocytes. J. Cell. Physiol. 220: 756–764, 2009.

Serine Racemase Suppresses Chondrogenic Differentiation in Cartilage in A Sox9-Dependent Manner

Serine racemase (SR) is responsible for the biosynthesis of D‐serine (D‐Ser), an endogenous co‐agonist for N‐methyl‐D‐aspartate (NMDA) receptors, from L‐serine (L‐Ser) in the central nervous system. In the present study, we investigated the role of SR in the regulation of chondrogenic differentiation in cartilage. On in situ hybridization analysis of tibia from neonatal rats, SR mRNA was ubiquitously expressed in all cell layers of proliferating to hypertrophic chondrocytes. In the pre‐chondrogenic cell line ATDC5 cells, mRNA expression was seen with SR irrespective of the cellular maturity, with no mRNA expression of the NR1 subunit essential for the heteromeric assembly of functional NMDA receptor channels. In ATDC5 cells stably overexpressing SR, significant inhibition was found with the maturation‐dependent temporal increases in Alcian blue staining, alkaline phosphatase (ALP) activity and mRNA expression of type II and type X collagens. Stable overexpression of SR significantly impaired the sry‐type HMG box 9 (Sox9) transcriptinal activity in ATDC5 cells, while Sox9 transcriptional activity was significantly inhibited in COS7 cells with co‐introduction of SR and Sox9. However, no significant inhibition was seen with Sox9 transcriptional activity in COS7 cells co‐introduced of either SRK56G defective of D‐Ser formation ability or 3‐phosphoglycerate dehydrogenase essential for D‐Ser biosynthesis. The co‐introduction of SR with Sox9 significantly decreased the Sox9 protein level with that of Sox9 mRNA being unchanged. These results suggest that SR may negatively regulate cellular differentiation through the inhibition of Sox9 transcriptional activity in chondrocytes. J. Cell. Physiol. 215: 320–328, 2008.

The transcription factor Foxc1 is necessary for Ihh–Gli2-regulated endochondral ossification

Indian hedgehog (Ihh) regulates endochondral ossification in both a parathyroid hormone-related protein (PTHrP)-dependent and -independent manner by activating transcriptional mediator Gli2. However, the molecular mechanisms underlying these processes remain elusive. Here by using in vivo microarray analysis, we identify forkhead box C1 (Foxc1) as a transcriptional partner of Gli2. Foxc1 stimulates expression of Ihh target genes, including PTHrP and Col10a1, through its physical and functional interaction with Gli2. Conversely, a dominant negative Foxc1 inhibits the Ihh target gene expression. In a spontaneous loss of Foxc1 function mouse (Foxc1(ch/ch)), endochondral ossification is delayed and the expression of Ihh target genes inhibited. Moreover, the pathological Foxc1 missense mutation observed in the Axenfeld-Rieger syndrome impairs Gli2-Foxc1 association as well as Ihh function. Our findings suggest that Foxc1 is an important transcriptional partner of Ihh-Gli2 signalling during endochondral ossification, and that disruption of the Foxc1-Gli2 interaction causes skeletal abnormalities observed in the Axenfeld-Rieger syndrome.

The transcription factor paired box-5 promotes osteoblastogenesis through direct induction of Osterix and Osteocalcin.

Although skeletal abnormalities are seen in mice deficient of particular paired box (Pax) family proteins, little attention has been paid to their role in osteoblastogenesis so far. Here, we investigated the possible involvement of several Pax family members in mechanisms underlying the regulation of differentiation and maturation of osteoblasts. Among different Pax family members tested, Pax5 was not markedly expressed in murine calvarial osteoblasts before culture, but progressively expressed by osteoblasts under differentiation toward maturation. Immunoreactive Pax5 was highly detectable in primary cultured mature osteoblasts on immunoblotting and in osteoblastic cells attached to cancellous bone in mouse tibial sections on immunohistochemistry, respectively. Knockdown by small interfering RNA (siRNA) of endogenous Pax5 led to significant inhibition of the expression of Osteocalcin, and Osterix through deterioration of gene transactivation, in addition to a1(I)Collagen expression and alkaline phosphatase (ALP) staining, without affecting runt‐related transcription factor‐2 (Runx2) expression and cell viability in osteoblastic MC3T3‐E1 cells. The introduction of Pax5 enhanced promoter activities of Osteocalcin and Osterix in a manner dependent on the paired domain in MC3T3‐E1 cells. Putative Pax5 binding sites were identified in the 5′‐flanking regions of mouse Osteocalcin and Osterix, whereas chromatin immunoprecipitation assay revealed the direct binding of Pax5 to particular regions of Osteocalcin and Osterix promoters in MC3T3‐E1 cells. Overexpression of Pax5 significantly increased Osteocalcin, Osterix, and a1(I)Collagen expression, ALP activity, and Ca2+ accumulation, without affecting Runx2 expression, in MC3T3‐E1 cells. In vertebrae of transgenic mice predominantly expressing Pax5 in osteoblasts, a significant increase was seen in the ratio of bone volume over tissue volume and the bone formation rate. These findings suggest that Pax5 could positively regulate osteoblastic differentiation toward maturation in vitro, in addition to promoting bone formation and remodeling in vivo, as one of the transcription factors essential for controlling osteoblastogenesis independently of Runx2.

Positive Regulation by GABABR1 Subunit of Leptin Expression through Gene Transactivation in Adipocytes

Background The view that γ-aminobutyric acid (GABA) plays a functional role in non-neuronal tissues, in addition to an inhibitory neurotransmitter role in the mammalian central nervous system, is prevailing, while little attention has been paid to GABAergic signaling machineries expressed by adipocytes to date. In this study, we attempted to demonstrate the possible functional expression of GABAergic signaling machineries by adipocytes. Methodology/Principal Findings GABAB receptor 1 (GABABR1) subunit was constitutively expressed by mouse embryonic fibroblasts differentiated into adipocytes and adipocytic 3T3-L1 cells in culture, as well as mouse white adipose tissue, with no responsiveness to GABABR ligands. However, no prominent expression was seen with mRNA for GABABR2 subunit required for heteromeric orchestration of the functional GABABR by any adipocytic cells and tissues. Leptin mRNA expression was significantly and selectively decreased in adipose tissue and embryonic fibroblasts, along with drastically reduced plasma leptin levels, in GABABR1-null mice than in wild-type mice. Knockdown by siRNA of GABABR1 subunit led to significant decreases in leptin promoter activity and leptin mRNA levels in 3T3-L1 cells. Conclusions/Significance Our results indicate that GABABR1 subunit is constitutively expressed by adipocytes to primarily regulate leptin expression at the transcriptional level through a mechanism not relevant to the function as a partner of heterodimeric assembly to the functional GABABR.