Masaki Ishikawa

Pannexin 3 functions as an ER Ca2+ channel, hemichannel, and gap junction to promote osteoblast differentiation

Pannexin 3 functions as an essential protein for Ca2+ and ATP transport and cell–cell communication during osteoblast differentiation

Pannexin 3 Regulates Intracellular ATP/cAMP Levels and Promotes Chondrocyte Differentiation *□

Pannexin 3 (Panx3) is a new member of the gap junction pannexin family, but its expression profiles and physiological function are not yet clear. We demonstrate in this study that Panx3 is expressed in cartilage and regulates chondrocyte proliferation and differentiation. Panx3 mRNA was expressed in the prehypertrophic zone in the developing growth plate and was induced during the differentiation of chondrogenic ATDC5 and N1511 cells. Panx3-transfected ATDC5 and N1511 cells promoted chondrogenic differentiation, but the suppression of endogenous Panx3 inhibited differentiation of ATDC5 cells and primary chondrocytes. Panx3-transfected ATDC5 cells reduced parathyroid hormone-induced cell proliferation and promoted the release of ATP into the extracellular space, possibly by action of Panx3 as a hemichannel. Panx3 expression in ATDC5 cells reduced intracellular cAMP levels and the activation of cAMP-response element-binding, a protein kinase A downstream effector. These Panx3 activities were blocked by anti-Panx3 antibody. Our results suggest that Panx3 functions to switch the chondrocyte cell fate from proliferation to differentiation by regulating the intracellular ATP/cAMP levels.

Plasma and Serum Lipidomics of Healthy White Adults Shows Characteristic Profiles by Subjects’ Gender and Age

Blood is a commonly used biofluid for biomarker discovery. Although blood lipid metabolites are considered to be potential biomarker candidates, their fundamental properties are not well characterized. We aimed to (1) investigate the matrix type (serum vs. plasma) that may be preferable for lipid biomarker exploration, (2) elucidate age- and gender-associated differences in lipid metabolite levels, and (3) examine the stability of lipid metabolites in matrix samples subjected to repeated freeze-thaw cycles. Using liquid chromatography-mass spectrometry, we performed lipidomic analyses for fasting plasma and serum samples for four groups (15 subjects/group) of young and elderly (25–34 and 55–64 years old, respectively) males and females and for an additional aliquot of samples from young males, which were subjected to repeated freeze-thaw cycles. Lysophosphatidylcholine and diacylglycerol levels were higher in serum than in plasma samples, suggesting that the clotting process influences serum lipid metabolite levels. Gender-associated differences highlighted that the levels of many sphingomyelin species were significantly higher in females than in males, irrespective of age and matrix (plasma and serum). Age-associated differences were more prominent in females than in males, and in both matrices, levels of many triacylglycerols were significantly higher in elderly females than in young females. Plasma and serum levels of most lipid metabolites were reduced by freeze-thawing. Our results indicate that plasma is an optimal matrix for exploring lipid biomarkers because it represents the original properties of an individual’s blood sample. In addition, the levels of some blood lipid species of healthy adults showed gender- and age-associated differences; thus, this should be considered during biomarker exploration and its application in diagnostics. Our fundamental findings on sample selection and handling procedures for measuring blood lipid metabolites is important for ensuring the quality of biomarkers identified and its qualification process.

Role of Epithelial-Stem Cell Interactions during Dental Cell Differentiation

Background: The role of dental epithelium in stem cell differentiation has not been clearly elucidated. Results: SP cells differentiated into odontoblasts by epithelial BMP4, whereas iPS cells differentiated into ameloblasts when cultured with dental epithelium. Conclusion: Stem cells can be induced to odontogenic cell fates when co-cultured with dental epithelium. Significance: This is the first report to show induction of ameloblasts from iPS cells. Epithelial-mesenchymal interactions regulate the growth and morphogenesis of ectodermal organs such as teeth. Dental pulp stem cells (DPSCs) are a part of dental mesenchyme, derived from the cranial neural crest, and differentiate into dentin forming odontoblasts. However, the interactions between DPSCs and epithelium have not been clearly elucidated. In this study, we established a mouse dental pulp stem cell line (SP) comprised of enriched side population cells that displayed a multipotent capacity to differentiate into odontogenic, osteogenic, adipogenic, and neurogenic cells. We also analyzed the interactions between SP cells and cells from the rat dental epithelial SF2 line. When cultured with SF2 cells, SP cells differentiated into odontoblasts that expressed dentin sialophosphoprotein. This differentiation was regulated by BMP2 and BMP4, and inhibited by the BMP antagonist Noggin. We also found that mouse iPS cells cultured with mitomycin C-treated SF2-24 cells displayed an epithelial cell-like morphology. Those cells expressed the epithelial cell markers p63 and cytokeratin-14, and the ameloblast markers ameloblastin and enamelin, whereas they did not express the endodermal cell marker Gata6 or mesodermal cell marker brachyury. This is the first report of differentiation of iPS cells into ameloblasts via interactions with dental epithelium. Co-culturing with dental epithelial cells appears to induce stem cell differentiation that favors an odontogenic cell fate, which may be a useful approach for tooth bioengineering strategies.

Critical roles of the TGF-β type I receptor ALK5 in perichondrial formation and function, cartilage integrity, and osteoblast differentiation during growth plate development

TGF-beta has been implicated in the proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-beta in skeletal development is unclear. In this study, we investigated the role of TGF-beta signaling in growth plate development by creating mice with a conditional knockout of the TGF-beta type I receptor ALK5 (ALK5(CKO)) in skeletal progenitor cells using Dermo1-Cre mice. ALK5(CKO) mice had short and wide long bones, reduced bone collars, and trabecular bones. In ALK5(CKO) growth plates, chondrocytes proliferated and differentiated, but ectopic cartilaginous tissues protruded into the perichondrium. In normal growth plates, ALK5 protein was strongly expressed in perichondrial progenitor cells for osteoblasts, and in a thin chondrocyte layer located adjacent to the perichondrium in the peripheral cartilage. ALK5(CKO) growth plates had an abnormally thin perichondrial cell layer and reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions. Using tamoxifen-inducible Cre-ER-mediated ALK5-deficient primary calvarial cell cultures, we found that TGF-beta signaling promoted osteoprogenitor proliferation, early differentiation, and commitment to the osteoblastic lineage through the selective MAPKs and Smad2/3 pathways. These results demonstrate the important roles of TGF-beta signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development.

Pannexin 3 Inhibits Proliferation of Osteoprogenitor Cells by Regulating Wnt and p21 Signaling

Background: The mechanism of the transition from osteoprogenitor cell proliferation to differentiation is unclear. Results: Panx3 inhibits osteoprogenitor proliferation by blocking canonical Wnt signaling and promoting p21 activation. Conclusion: A Panx3 hemichannel induces multiple Panx3 signaling pathways critical for the cell cycle exit. Significance: Our findings reveal that Panx3 is a new regulator to switch the stage from proliferation to differentiation in osteoprogenitor cells. Canonical Wnt signaling and BMP promote the proliferation and differentiation of osteoprogenitors, respectively. However, the regulatory mechanism involved in the transition from proliferation to differentiation is unclear. Here, we show that Panx3 (pannexin 3) plays a key role in this transition by inhibiting the proliferation and promoting the cell cycle exit. Using primary calvarial cells and explants, C3H10T1/2 cells, and C2C12 cells, we found that Panx3 expression inhibited cell growth, whereas the inhibition of endogenous Panx3 expression increased it. We also found that the Panx3 hemichannel inhibited cell growth by promoting β-catenin degradation through GSK3β activation. Additionally, the Panx3 hemichannel inhibited cyclin D1 transcription and Rb phosphorylation through reduced cAMP/PKA/CREB signaling. Furthermore, the Panx3 endoplasmic reticulum Ca2+ channel induced the transcription and phosphorylation of p21, through the calmodulin/Smad pathway, and resulted in the cell cycle exit. Our results reveal that Panx3 is a new regulator that promotes the switch from proliferation to differentiation of osteoprogenitors via multiple Panx3 signaling pathways.

The DNA-binding inhibitor Id3 regulates IL-9 production in CD4(+) T cells.

The molecular mechanisms by which signaling via transforming growth factor-β (TGF-β) and interleukin 4 (IL-4) control the differentiation of CD4+ IL-9-producing helper T cells (TH9 cells) remain incompletely understood. We found here that the DNA-binding inhibitor Id3 regulated TH9 differentiation, as deletion of Id3 increased IL-9 production from CD4+ T cells. Mechanistically, TGF-β1 and IL-4 downregulated Id3 expression, and this process required the kinase TAK1. A reduction in Id3 expression enhanced binding of the transcription factors E2A and GATA-3 to the Il9 promoter region, which promoted Il9 transcription. Notably, Id3-mediated control of TH9 differentiation regulated anti-tumor immunity in an experimental melanoma-bearing model in vivo and also in human CD4+ T cells in vitro. Thus, our study reveals a previously unrecognized TAK1–Id3–E2A–GATA-3 pathway that regulates TH9 differentiation.

PARP-1 regulates expression of TGF-β receptors in T cells

Transforming growth factor-β (TGF-β) receptors (TβRs) are essential components for TGF-β signal transduction in T cells, yet the mechanisms by which the receptors are regulated remain poorly understood. We show here that Poly(ADP-ribose) polymerase-1 (PARP-1) regulates TGF-β receptor I (TβRI) and II (TβRII) expression in CD4(+) T cells and subsequently affects Smad2/3-mediated TGF-β signal transduction. Inhibition of PARP-1 led to the upregulation of both TβRI and TβRII, yet the underlying molecular mechanisms were distinct. PARP-1 selectively bound to the promoter of TβRII, whereas the enzymatic activity of PARP-1 was responsible for the inhibition of TβRI expression. Importantly, inhibition of PARP-1 also enhanced expression of TβRs in human CD4(+) T cells. Thus, PARP-1 regulates TβR expression and TGF-β signaling in T cells.

Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta

Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50–70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.

Pannexin 3 and connexin 43 modulate skeletal development through their distinct functions and expression patterns

ABSTRACT Pannexin 3 (Panx3) and connexin 43 (Cx43; also known as GJA1) are two major gap junction proteins expressed in osteoblasts. Here, we studied their functional relationships in skeletal formation by generating Panx3−/− and Panx3−/−;Cx43−/− mice and comparing their skeletal phenotypes with Cx43−/− mice. Panx3−/− mice displayed defects in endochondral and intramembranous ossification, resulting in severe dwarfism and reduced bone density. The skeletal abnormalities of Panx3−/−;Cx43−/− mice were similar to those in Panx3−/− mice. The gross appearance of newborn Cx43−/− skeletons showed no obvious abnormalities, except for less mineralization of the skull. In Panx3−/− mice, proliferation of chondrocytes and osteoblasts increased and differentiation of these cells was inhibited. Panx3 promoted expression of osteogenic proteins such as ALP and Ocn (also known as ALPL and BGLAP, respectively), as well as Cx43, by regulating Osx (also known as SP7) expression. Panx3 was induced in the early differentiation stage and reduced during the maturation stage of osteoblasts, when Cx43 expression increased in order to promote mineralization. Furthermore, only Panx3 functioned as an endoplasmic reticulum (ER) Ca2+ channel to promote differentiation, and it could rescue mineralization defects in Cx43−/− calvarial cells. Our findings reveal that Panx3 and Cx43 have distinct functions in skeletal formation. Summary: Panx3 and Cx43 are two important gap junction proteins expressed in osteoblasts. We find that Panx3 and Cx43 regulate skeletal formation through their distinct expression patterns and functions.