Bile Acid Inhibition of N-type Calcium Channel Currents from Sympathetic Ganglion Neurons

Under some pathological conditions as bile flow obstruction or liver diseases with the enterohepatic circulation being disrupted, regurgitation of bile acids into the systemic circulation occurs and the plasma level of bile acids increases. Bile acids in circulation may affect the nervous system. We examined this possibility by studying the effects of bile acids on gating of neuronal (N)-type Ca2+ channel that is essential for neurotransmitter release at synapses of the peripheral and central nervous system. N-type Ca2+ channel currents were recorded from bullfrog sympathetic neuron under a cell-attached mode using 100 mM Ba2+ as a charge carrier. Cholic acid (CA, 10-6 M) that is relatively hydrophilic thus less cytotoxic was included in the pipette solution. CA suppressed the open probability of N-type Ca2+ channel, which appeared to be due to an increase in null (no activity) sweeps. For example, the proportion of null sweep in the presence of CA was ~40% at +40 mV as compared with ~8% in the control recorded without CA. Other single channel properties including slope conductance, single channel current amplitude, open and shut times were not significantly affected by CA being present. The results suggest that CA could modulate N-type Ca2+ channel gating at a concentration as low as 10-6 M. Bile acids have been shown to activate nonselective cation conductance and depolarize the cell membrane. Under pathological conditions with increased circulating bile acids, CA suppression of N-type Ca2+ channel function may be beneficial against overexcitation of the synapses

Crosstalk between Nuclear Factor I-C and Transforming Growth Factor-β1 Signaling Regulates Odontoblast Differentiation and Homeostasis

Transforming growth factor-β1 (TGF-β1) signaling plays a key role in vertebrate development, homeostasis, and disease. Nuclear factor I-C (NFI-C) has been implicated in TGF-β1 signaling, extracellular matrix gene transcription, and tooth root development. However, the functional relationship between NFI-C and TGF-β1 signaling remains uncharacterized. The purpose of this study was to identify the molecular interactions between NFI-C and TGF-β1 signaling in mouse odontoblasts. Real-time polymerase chain reaction and western analysis demonstrated that NFI-C expression levels were inversely proportional to levels of TGF-β1 signaling molecules during in vitro odontoblast differentiation. Western blot and immunofluorescence results showed that NFI-C was significantly degraded after TGF-β1 addition in odontoblasts, and the formation of the Smad3 complex was essential for NFI-C degradation. Additionally, ubiquitination assay results showed that Smurf1 and Smurf2 induced NFI-C degradation and polyubiquitination in a TGF-β1-dependent manner. Both kinase and in vitro binding assays revealed that the interaction between NFI-C and Smurf1/Smurf2 requires the activation of the mitogen-activated protein kinase pathway by TGF-β1. Moreover, degradation of NFI-C induced by TGF-β1 occurred generally in cell types other than odontoblasts in normal human breast epithelial cells. In contrast, NFI-C induced dephosphorylation of p-Smad2/3. These results show that crosstalk between NFI-C and TGF-β1 signaling regulates cell differentiation and homeostatic processes in odontoblasts, which might constitute a common cellular mechanism

Notum regulates the cusp and root patterns in mouse molar

Abstract Notum is a direct target of Wnt/β-catenin signaling and plays a crucial role as a Wnt inhibitor within a negative feedback loop. In the tooth, Notum is known to be expressed in odontoblasts, and severe dentin defects and irregular tooth roots have been reported in Notum-deficient mice. However, the precise expression pattern of Notum in early tooth development, and the role of Notum in crown and root patterns remain elusive. In the present study, we identified a novel Notum expression in primary enamel knot (EK), secondary EKs, and dental papilla during tooth development. Notum-deficient mice exhibited enlarged secondary EKs, resulting in broader cusp tips, altered cusp patterns, and reduced concavity in crown outline. These alterations in crown outline led to a reduction in cervical tongue length, thereby inducing root fusion in Notum-deficient mice. Overall, these results suggest that the secondary EK size, regulated by the Wnt/Notum negative feedback loop, has a significant impact on the patterns of crown and root during tooth morphogenesis

BST2 Mediates Osteoblast Differentiation via the BMP2 Signaling Pathway in Human Alveolar-Derived Bone Marrow Stromal Cells.

The molecular mechanisms controlling the differentiation of bone marrow stromal stem cells into osteoblasts remain largely unknown. In this study, we investigated whether bone marrow stromal antigen 2 (BST2) influences differentiation toward the osteoblasts lineage. BST2 mRNA expression in human alveolar-derived bone marrow stromal cells (hAD-BMSCs) increased during differentiation into osteoblasts. hAD-BMSCs differentiation into osteoblasts and the mRNA expression of the bone-specific markers alkaline phosphatase, collagen type α 1, bone sialoprotein, osteocalcin, and osterix were reduced by BST2 knockdown using siRNA. Furthermore, BST2 knockdown in hAD-BMSCs resulted in decreased RUNX2 mRNA and protein expression. We hypothesized that BST2 is involved in differentiation of into osteoblasts via the BMP2 signaling pathway. Accordingly, we evaluated the mRNA expression levels of BMP2, BMP receptors (BMPR1 and 2), and the downstream signaling molecules SMAD1, SMAD4, and p-SMAD1/5/8 in BST2 knockdown cells. BMP2 expression following the induction of differentiation was significantly lower in BST2 knockdown cells than in cells treated with a non-targeting control siRNA. Similar results were found for the knockdown of the BMP2 receptor- BMPR1A. We also identified significantly lower expression of SMAD1, SMAD4, and p-SMAD1/5/8 in the BST2 knockdown cells than control cells. Our data provide the first evidence that BST2 is involved in the osteogenic differentiation of bone marrow stromal cells via the regulation of the BMP2 signaling pathway

Fibroblast growth factor-4 enhances proliferation of mouse embryonic stem cells via activation of c-Jun signaling.

Fibroblast growth factor-4 (FGF4) is expressed in embryonic stages and in adult tissues, where it plays critical roles in modulating multiple cellular functions. However, the exact roles of FGF4 on proliferation and differentiation of embryonic stem cells (ESCs) are not completely understood. Exogenous addition of FGF4 stimulated proliferation of mouse ESCs (mESCs), as proven by the increases in DNA synthesis and cell cycle regulatory protein induction. These increases were almost completely inhibited by pre-treating cells with anti-FGF4 antibody. FGF4 also activated c-Jun N-terminal kinase (JNK) and extracellular-signal regulated kinase (ERK) signaling, but not p38 kinase. Blockage of JNK signaling by SP600125 or by transfection with its specific siRNA significantly inhibited FGF4-stimulated cell proliferation through the suppression of c-Jun induction and activator protein-1 (AP-1) activity. However, ERK or p38 kinase inhibitor did not affect FGF4-stimulated proliferation in mESCs. FGF4 suppressed osteogenic differentiation of mESCs by inhibiting expression of transcription factors involved in bone formation. Further, exogenous FGF4 addition stimulated proliferation of human periodontal ligament stem cells (hPDLSCs) and bone marrow mesenchymal stem cells (BMMSCs) via activation of ERK signaling. FGF4 also augmented mineralization of hPDLSCs, but not of BMMSCs. Collectively, it is suggested that FGF4 triggers proliferation of stem cells by activating MAPK-mediated signaling, while it affects differently osteogenic differentiation according to the origins of stem cells