The biphasic effect of extracellular glucose concentration on carbachol-induced fluid secretion from mouse submandibular glands.

Cholinergic agonists evoke elevations of the cytoplasmic free-calcium concentration ([Ca2+ ]i ) to stimulate fluid secretion in salivary glands. Salivary flow rates are significantly reduced in diabetic patients. However, it remains elusive how salivary secretion is impaired in diabetes. Here, we used an ex vivo submandibular gland perfusion technique to characterize the dependency of salivary flow rates on extracellular glucose concentration and activities of glucose transporters expressed in the glands. The cholinergic agonist carbachol (CCh) induced sustained fluid secretion, the rates of which were modulated by the extracellular glucose concentration in a biphasic manner. Both lowering the extracellular glucose concentration to less than 2.5 mM and elevating it to higher than 5 mM resulted in decreased CCh-induced fluid secretion. The CCh-induced salivary flow was suppressed by phlorizin, an inhibitor of the sodium-glucose cotransporter 1 (SGLT1) located basolaterally in submandibular acinar cells, which is altered at the protein expression level in diabetic animal models. Our data suggest that SGLT1-mediated glucose uptake in acinar cells is required to maintain the fluid secretion by sustaining Cl- secretion in real-time. High extracellular glucose levels may suppress the CCh-induced secretion of salivary fluid by altering the activities of ion channels and transporters downstream of [Ca2+ ]i signals.

Different rate-limiting activities of intracellular pH regulators for HCO3- secretion stimulated by forskolin and carbachol in rat parotid intralobular ducts.

Intracellular pH (pHi) regulation fundamentally participates in maintaining HCO3- release from HCO3--secreting epithelia. We used parotid intralobular ducts loaded with BCECF to investigate the contributions of a carbonic anhydrase (CA), anion channels and a Na+-H+ exchanger (NHE) to pHi regulation for HCO3- secretion by cAMP and Ca2+ signals. Resting pHi was dispersed between 7.4 and 7.9. Forskolin consistently decreased pHi showing the dominance of pHi-lowering activities, but carbachol gathered pHi around 7.6. CA inhibition suppressed the forskolin-induced decrease in pHi, while it allowed carbachol to consistently increase pHi by revealing that carbachol prominently activated NHE via Ca2+-calmodulin. Under NHE inhibition, forskolin and carbachol induced the remarkable decreases in pHi, which were slowed predominantly by CA inhibition and by CA or anion channel inhibition, respectively. Our results suggest that forskolin and carbachol primarily activate the pHi-lowering CA and pHi-raising NHE, respectively, to regulate pHi for HCO3- secretion.

Phf14, a novel regulator of mesenchyme growth via platelet-derived growth factor (PDGF) receptor-α.

The regulation of mesenchymal cell growth by signaling molecules plays an important role in maintaining tissue functions. Aberrant mesenchymal cell proliferation caused by disruption of this regulatory process leads to pathogenetic events such as fibrosis. In the current study we have identified a novel nuclear factor, Phf14, which controls the proliferation of mesenchymal cells by regulating PDGFRα expression. Phf14-null mice died just after birth due to respiratory failure. Histological analyses of the lungs of these mice showed interstitial hyperplasia with an increased number of PDGFRα(+) mesenchymal cells. PDGFRα expression was elevated in Phf14-null mesenchymal fibroblasts, resulting in increased proliferation. We demonstrated that Phf14 acts as a transcription factor that directly represses PDGFRα expression. Based on these results, we used an antibody against PDGFRα to successfully treat mouse lung fibrosis. This study shows that Phf14 acts as a negative regulator of PDGFRα expression in mesenchymal cells undergoing normal and abnormal proliferation, and is a potential target for new treatments of lung fibrosis.

Differentiation of mesodermal cells from pluripotent stem cells.

The pluripotency of embryonic stem cells has been well demonstrated by a vast variety of studies showing the induction of differentiation into desired cell types that have the potential to be used not only in basic studies but also in medical applications. The induction of mesodermal cells, especially blood cells, from embryonic stem cells is notable from the point of view of transplantation, and the methods for this induction have improved over the last few years, with more defined culture conditions in place. Concurrently, the generation of induced pluripotent stem cells from somatic cells opens the possibility of autologous transplantation. In fact, there are a growing number of reports demonstrating that several mesodermal cells can be differentiated from induced pluripotent stem cells using the same methods used for embryonic stem cells. This review summarizes recent advances in the differentiation of mesodermal cells from embryonic stem cells and induced pluripotent stem cells.

A Sveinsson's chorioretinal atrophy-associated missense mutation in mouse Tead1 affects its interaction with the co-factors YAP and TAZ.

Sveinsson's chorioretinal atrophy (SCRA) is an autosomal dominant eye disease characterized by bilateral chorioretinal degeneration. A missense mutation in the gene encoding the transcription factor TEAD1/TEF-1 (Y421H) is genetically linked to SCRA, but the mechanisms of pathology remain unclear. To study the molecular mechanisms underlying SCRA, a missense mutation corresponding to Y421H in human TEAD1 was introduced into mouse Tead1 (Y410H), and a functional analysis of the mutant protein was performed in RPE-J cells. The missense mutation reduced the ability of Tead1 to interact with the co-factors YAP and TAZ, but not with the co-factors Vgl-1, -2, and -3, in a mammalian two-hybrid assay. A GST pull-down assay showed that the direct interaction between Tead1 and YAP or TAZ was lost owing to the mutation. Amino acid substitutions at position 410 of Tead1 revealed the essentiality of this tyrosine residue to the interaction. The Y410H mutation also abolished the transcriptional activity of Tead1 under the co-expression of YAP or TAZ. These results suggest that SCRA pathogenesis may be due to a loss-of-function of TEAD1 affecting the regulation of its target genes.

Microarray analysis of the genes induced by tetracycline-regulated expression of NDRF/NeuroD2 in P19 cells.

NeuroD-related factor (NDRF)/NeuroD2 is a basic helix-loop-helix (bHLH) protein that plays important roles in neuronal development. To elucidate the NDRF transcription network, we used mouse cDNA microarray analysis combined with a tetracycline-regulatable expression system in P19 embryonal carcinoma cells. Five genes were identified to be up-regulated in the presence of NDRF protein. RNA hybridization analysis confirmed that brain-lipid-binding protein (BLBP) and inhibitor of differentiation 1 (Id1) genes were among the five genes that were rapidly and significantly up-regulated after induction of NDRF. When a dominant negative form of NDRF protein was expressed during retinoic acid-induced neuronal differentiation of P19 cells, the BLBP gene, but not the Id1 gene, was potently repressed. Immunohistochemical analysis revealed that both NDRF and Id1 immunoreactivities were observed in some granule cells of the cerebellum in the postnatal period. These results suggest that NDRF or its related bHLH proteins may act upstream of these genes in a subset of developing neurons.

Characterization of novel splicing variants of the mouse MCF-2 (DBL) proto-oncogene.

MCF-2 (DBL) proto-oncogene is a prototype guanine nucleotide exchange factor (GEF) that modulates Rho GTPases such as Rho, Rac, and Cdc42. Although the partial sequence of mouse MCF-2 has been determined, its full-length cDNA and biochemical functions had not been elucidated. We isolated the complete mouse MCF-2 cDNA and obtained recombinant functional protein. Homology between the mouse and human MCF-2 (DBL) cDNAs is 75.08% identity and between the mouse and human amino acid sequences 74.52% identity. Analysis of tissue distribution showed that mouse MCF-2 mRNA is expressed in brain, kidney, intestine, and testis. The brain-specific transcript is an alternatively spliced derivative that omits the 48bp exon 11. A similar alternatively spliced mRNA product is also found in humans (DBL). Guanine nucleotide exchange activities of the testis-expressed mouse Mcf-2 and human Dbl were analyzed using RhoA, Rac1, and Cdc42 as substrates. RhoA and Cdc42 were activated similarly by both gene products, but Rac1 was activated only by the mouse product. The brain-specific Mcf-2 gene product, and its human counterpart, was less active than the respective testis-specific products. This indicates that the element encoded by the 48bp exon missing in the brain transcripts is necessary for full GEF activity. This report provides fundamental data on the structure of Mcf-2, which regulates a variety of cellular signaling pathways.

Alternative splicing variants of the human DBL (MCF-2) proto-oncogene.

The DBL (MCF-2) proto-oncogene is a prototype guanine nucleotide exchange factor (GEF) that modulates the Rho family of GTPases. In this communication we describe the isolation of three novel splicing variants of Dbl. The prototype Dbl gene (designated var.1 here) contains 25 exons, while splicing variant 2 (var.2) lacks exons 23 and 24. Var.3 contains additional 3 exons from 5(')-UTR in place of exon 1, while var.4, var.2, and var.3 contain a 48bp insertion between exons 10 and 11, resulting in the insertion of 16 amino acids. We found that var.1 was expressed only in brain, whereas var.3 was expressed in heart, kidney, spleen, liver, and testis, and var.4 in brain, heart, kidney, testis, placenta, stomach, and peripheral blood. The Dbl protein was detectable in brain, heart, kidney, intestine, muscle, lung, and testis. An assay for GEF activity revealed that the var.2 exhibits decreased GEF activity towards Cdc42, var.3 exhibits a weak but significant activity toward Rac1 and Cdc42, var.4 exhibits significant activity toward RhoA and Cdc42, while var.1 exhibits no activity toward RhoA, Rac1, or Cdc42. In summary, we describe 4 splicing variants of the human DBL proto-oncogene that show different tissue distributions and GEF specificities.