Next-generation sequencing of patients with congenital anosmia.

We performed whole exome or genome sequencing in eight multiply affected families with ostensibly isolated congenital anosmia. Hypothesis-free analyses based on the assumption of fully penetrant recessive/dominant/X-linked models obtained no strong single candidate variant in any of these families. In total, these eight families showed 548 rare segregating variants that were predicted to be damaging, in 510 genes. Three Kallmann syndrome genes (FGFR1, SEMA3A, and CHD7) were identified. We performed permutation-based analysis to test for overall enrichment of these 510 genes carrying these 548 variants with genes mutated in Kallmann syndrome and with a control set of genes mutated in hypogonadotrophic hypogonadism without anosmia. The variants were found to be enriched for Kallmann syndrome genes (3 observed vs. 0.398 expected, p = 0.007), but not for the second set of genes. Among these three variants, two have been already reported in genes related to syndromic anosmia (FGFR1 (p.(R250W)), CHD7 (p.(L2806V))) and one was novel (SEMA3A (p.(T717I))). To replicate these findings, we performed targeted sequencing of 16 genes involved in Kallmann syndrome and hypogonadotrophic hypogonadism in 29 additional families, mostly singletons. This yielded an additional 6 variants in 5 Kallmann syndrome genes (PROKR2, SEMA3A, CHD7, PROK2, ANOS1), two of them already reported to cause Kallmann syndrome. In all, our study suggests involvement of 6 syndromic Kallmann genes in isolated anosmia. Further, we report a yet unreported appearance of di-genic inheritance in a family with congenital isolated anosmia. These results are consistent with a complex molecular basis of congenital anosmia.

Induction of the SHARP-2 mRNA level by insulin is mediated by multiple signaling pathways.

The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7 kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.

(-)-Epigallocatechin-3-gallate stimulates both AMP-activated protein kinase and nuclear factor-kappa B signaling pathways.

We previously reported that (-)-epigallocatechin-3-gallate (EGCG) increased the level of SHARP-1 mRNA via a phosphoinositide 3-kinase/atypical protein kinase C lambda signaling pathway in rat H4IIE hepatoma cells. In the present study, we investigated other signaling pathway(s). Treating with either compound-C, BAY11-7082, or both, partially blocked the up-regulation of the SHARP-1 gene by EGCG. This suggests that AMP-activated protein kinase (AMPK)- and nuclear factor-kappa B (NF-κB)-signaling pathways were additively involved in the induction mediated by EGCG. Indeed, an AMPK activator induced a level of SHARP-1 mRNA. Although actinomycin D partially blocked the EGCG-induction of that SHARP-1 mRNA level, the nucleotide sequence between -1501 and -1 in the rat SHARP-1 gene did not positively respond to EGCG and NF-κB, respectively. Thus, we conclude that EGCG stimulates multiple signaling pathways in the SHARP-1 gene expression at the transcriptional and post-transcriptional levels and that there is no regulatory region susceptible to EGCG and NF-κB in the examined region.

Analysis of induction mechanisms of an insulin-inducible transcription factor SHARP-2 gene by (-)-epigallocatechin-3-gallate.

The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor. In this study, we examined the mechanism(s) involved in the regulation of the rat SHARP-2 gene expression by (-)-epigallocatechin-3-gallate (EGCG). The induction of SHARP-2 mRNA by EGCG was repressed by pretreatments with inhibitors for either phosphoinositide 3-kinase (PI3K) or RNA polymerase II. Then, we examined a biological relationship between EGCG and transcription factor NF-κB interfering with the insulin action. The protein levels of the NF-κB were rapidly decreased by an EGCG treatment. Finally, the mechanism(s) of transcriptional activation of the rat SHARP-2 gene by both NF-κB and EGCG was analyzed. While overexpression of the NF-κB p65 protein decreased the promoter activity of the SHARP-2 gene, EGCG did not affect it. Thus, we conclude that EGCG induces the expression of the rat SHARP-2 gene via both the PI3K pathway and degradation of the NF-κB p65 protein.

Analysis of regulatory mechanisms of an insulin-inducible SHARP-2 gene by (S)-Equol.

Small compounds that activate the insulin-dependent signaling pathway have potential therapeutic applications in controlling type 2 diabetes mellitus. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor that decreases expression of the phosphoenolpyruvate carboxykinase gene, a gluconeogenic enzyme gene. In this study, we screened for soybean isoflavones that can induce the rat SHARP-2 gene expression and analyzed their mechanism(s). Genistein and (S)-Equol, a metabolite of daidzein, induced rat SHARP-2 gene expression in H4IIE rat hepatoma cells. The (S)-Equol induction was mediated by both the phosphoinositide 3-kinase- and protein kinase C (PKC)-pathways. When a dominant negative form of atypical PKC lambda (aPKCλ) was expressed, the induction of SHARP-2 mRNA level by (S)-Equol was inhibited. In addition, Western blot analyses showed that (S)-Equol rapidly activated both aPKCλ and classical PKC alpha. Furthermore, the (S)-Equol induction was inhibited by treatment with a RNA polymerase inhibitor or a protein synthesis inhibitor. Finally, a reporter gene assay revealed that the transcriptional stimulation by (S)-Equol was mediated by nucleotide sequences located between -4687 and -4133 of the rat SHARP-2 gene. Thus, we conclude that (S)-Equol is an useful dietary supplement to control type 2 diabetes mellitus.

(-)-Epigallocatechin-3-gallate enhances the expression of an insulin-inducible transcription factor gene via a phosphoinositide 3-kinase/atypical protein kinase C lambda pathway.

The rat enhancer of split- and hairy-related protein-1 (SHARP-1) is an insulin-inducible transcriptional repressor. In this study, we examined issues of whether (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, regulates the expression of the rat SHARP-1 gene and which signaling pathway mediates the regulation. When H4IIE cells were treated with EGCG, SHARP-1 mRNA levels rapidly increased. Pretreatments with inhibitors for either phosphoinositide 3-kinase (PI 3-K) or protein kinase C partially blocked EGCG induction. Atypical protein kinase C lambda (aPKCλ) is known as a downstream target of PI 3-K in the liver. When a dominant-negative form of aPKCλ was expressed, the EGCG-induced SHARP-1 mRNAs was inhibited. Finally, Western blot analysis revealed that EGCG rapidly and temporarily stimulates aPKCλ phosphorylation. Thus, we conclude that EGCG induces SHARP-1 gene expression via a PI 3-K/aPKCλ signaling pathway.

Genistein stimulates the insulin-dependent signaling pathway.

Small compounds that activate the insulin-dependent signaling pathway have potential therapeutic applications in controlling insulin-independent diabetes mellitus. In this study, we investigated whether soybean isoflavones could induce the expression of SHARP-2, a downstream component of insulin-dependent signaling pathway, associated with the regulation of blood glucose. One such compound called genistein, rapidly and temporarily induced SHARP-2 mRNA levels in a dose-dependent manner in rat H4IIE hepatoma cells. This induction process was rapidly stimulated by a protein kinase C (PKC) activator and blocked by a PKC inhibitor, suggesting that SHARP-2 may be induced via PKC activation. Upon Western blot analysis, genistein showed a stimulation of PKC phosphorylation. Therefore, we concluded that genistein might transcriptionally induce SHARP-2 through the activation of PKC in H4IIE cells. Our results suggest that genistein might be a useful dietary supplement to control insulin-independent diabetes mellitus by inducing the SHARP-2 expression via a bypass of the insulin-dependent signaling pathway.

ZHX2 and ZHX3 repress cancer markers in normal hepatocytes.

ZHX2 and ZHX3 are the members of the ZHX transcriptional repressor family. To investigate the regulatory role of the repressors in hepatocytes and their involvement in carcinogenesis, the expression levels of ZHX2 and ZHX3 mRNAs were examined. The dRLh-84 hepatoma cells considerably expressed cancer marker genes PKM and HK II that are expressed in developing fetal tissues and cancer cells but repressed in normal hepatocytes. In dRLh-84 cells, the expression levels of ZHX2 and ZHX3 were very low compared with rat hepatocytes. Upon the reporter gene analysis utilizing the promoter region of these genes, ZHX3 repressed the transcription of the reporter luciferase gene from both promoters while ZHX2 only repressed that from HK II promoter. The promoter activity of alpha-fetoprotein was also repressed by the expression of ZHX2 in HLE hepatoma cells in a dose-dependent manner. We concluded that ZHX2 and ZHX3 were involved in the transcriptional repression of the hepatocellular cacinoma markers in normal hepatocytes, suggesting that the failure of the ZHX2 and/or ZHX3 expression might be a critical factor in the hepatocellular carcinogenesis.