Function of cytochrome P450 enzymes MycCI and MycG in Micromonospora griseorubida, a producer of the macrolide antibiotic mycinamicin.

The cytochrome P450 enzymes MycCI and MycG are encoded within the mycinamicin biosynthetic gene cluster and are involved in the biosynthesis of mycinamicin II (a 16-membered macrolide antibiotic produced by Micromonospora griseorubida). Based on recent enzymatic studies, MycCI is characterized as the C-21 methyl hydroxylase of mycinamicin VIII, while MycG is designated multifunctional P450, which catalyzes hydroxylation and also epoxidation at C-14 and C-12/13 on the macrolactone ring of mycinamicin. Here, we confirm the functions of MycCI and MycG in M. griseorubida. Protomycinolide IV and mycinamicin VIII accumulated in the culture broth of the mycCI disruption mutant; moreover, the mycCI gene fragment complemented the production of mycinamicin I and mycinamicin II, which are produced as major mycinamicins by the wild strain M. griseorubida A11725. The mycG disruption mutant did not produce mycinamicin I and mycinamicin II; however, mycinamicin IV accumulated in the culture broth. The mycG gene was located immediately downstream of the self-resistance gene myrB. The mycG gene under the control of mycGp complemented the production of mycinamicin I and mycinamicin II. Furthermore, the amount of mycinamicin II produced by the strain complemented with the mycG gene under the control of myrBp was approximately 2-fold higher than that produced by the wild strain. In M. griseorubida, MycG recognized mycinamicin IV, mycinamicin V, and also mycinamicin III as the substrates. Moreover, it catalyzed hydroxylation and also epoxidation at C-14 and C-12/13 on these intermediates. However, C-14 on mycinamicin I was not hydroxylated.

Gene targeting for O-methyltransferase genes, mycE and mycF, on the chromosome of Micromonospora griseorubida producing mycinamicin with a disruption cassette containing the bacteriophage phi C31 attB attachment site.

Mycinamicin, a 16-membered macrolide antibiotic produced by Micromonospora griseorubida, comprises a macrolactone and two deoxysugars: desosamine and mycinose. Mycinose is synthesized through two modification steps: the methylation of 6-deoxyallose in mycinamicin VI and of javose in mycinamicin III. To confirm the role of mycE and mycF genes in mycinamicin biosynthesis in M. griseorubida, disruption mutants of mycE and mycF were constructed by disruption plasmids containing attB in the disruption cassette FRT-neo-oriT-FRT-attB for the integration of phiC31-derivative vector plasmids; the disruption mutants were complemented through the integration of pSET152 derivatives containing intact mycE or mycF into the artificially inserted attB site. These disruption mutants did not produce mycinamicin II, but mainly accumulated mycinamicins VI and III, indicating that MycE and MycF methylated the C2''-OH group of 6-deoxyallose in mycinamicin VI and the C3''-OH group of C2''-methylated 6-deoxyallose in mycinamicin III, respectively. The complemented strains of mycE and mycF recovered the mycinamicin II productivity.

Relation of the expression of transcriptional factor TFAP2B to that of adipokines in subcutaneous and omental adipose tissues.

To determine the potential role of the transcriptional factor-activating enhancer-binding protein-2beta (TFAP2B) in the regulation of expression of adipokines, adiponectin, leptin, and interleukin-6 (IL-6) in vivo, we quantified the mRNA expression levels of these adipokines and TFAP2B in visceral (omental) and abdominal subcutaneous adipose tissues of 66 individuals with variable degree of adiposity and studied their correlations with BMI and their plasma concentrations. We found that BMI correlated negatively with plasma adiponectin levels and positively with those of leptin. Adiponection mRNA expression in subcutaneous fat correlated negatively with BMI, whereas leptin mRNA levels in the omentum correlated with plasma leptin levels and BMI. In contrast, IL-6 mRNA levels in subcutaneous and omental fat did not correlate with BMI. IL-6 mRNA levels in the omental fat correlated with plasma IL-6 levels. Whereas TFAP2B mRNA expression did not correlate with BMI, it correlated negatively with adiponectin expression in the subcutaneous adipose tissue. Furthermore, TFAP2B mRNA expression correlated negatively with leptin and positively with IL-6 expression in both subcutaneous and omental adipose tissues. These relationships are consistent with our in vitro observations and indicate that TFAP2B seems to regulate the expression of various adipokines in vivo.

Transcription factor AP-2beta inhibits glucose-induced insulin secretion in cultured insulin-secreting cell-line.

AIM: We previously identified the transcription factor activating enhancer-binding protein-2beta (AP-2beta) gene as a new candidate for conferring susceptibility to type 2 diabetes. To ascertain the possible involvement of AP-2beta in the pathogenesis of type 2 diabetes we examined the effects of AP-2beta on glucose-induced insulin secretion. METHODS: We measured the insulin secretion stimulated by glucose, tolbutamide, or KCl in the HIT-T15 cells infected with adenovirus vectors encoding AP-2beta or LacZ (control). RESULTS: We identified clear expression of AP-2beta in isolated rat pancreatic islets and in HIT-T15 cells. Glucose-induced increase in insulin secretion was significantly inhibited in AP-2beta-overexpressing cells (LacZ, 5.0+/-0.8 ng h(-1)mg(-1) protein; AP-2beta, 1.7+/-0.2 ng h(-1)mg(-1) protein; P=0.0015), whereas insulin expression was the same in both types of cells. Tolbutamide-induced insulin secretion was also suppressed in the AP-2beta-overexpressing cells, but KCl-induced insulin secretion was not affected by AP-2beta overexpression. In addition, Kir6.2 and glucokinase expression was significantly decreased in the AP-2beta-overexpressing cells. CONCLUSION: We identified for the first time that AP-2beta expressed and functioned in insulin-secreting cell-line HIT-T15. These results suggest that AP-2beta contributes to susceptibility to type 2 diabetes by inhibiting glucose-induced insulin secretion in pancreatic beta cells.

Production of rosamicin derivatives in Micromonospora rosaria by introduction of D-mycinose biosynthetic gene with PhiC31-derived integration vector pSET152.

Some of the polyketide-derived bioactive compounds contain sugars attached to the aglycone core, and these sugars often impart specific biological activity to the molecule or enhance this activity. Mycinamicin II, a 16-member macrolide antibiotic produced by Micromonospora griseorubida A11725, contains a branched lactone and two different deoxyhexose sugars, D-desosamine and D-mycinose, at the C-5 and C-21 positions, respectively. The D-mycinose biosynthesis genes, mycCI, mycCII, mycD, mycE, mycF, mydH, and mydI, present in the M. griseorubida A11725 chromosome were introduced into pSET152 under the regulation of the promoter of the apramycin-resistance gene aac(3)IV. The resulting plasmid pSETmycinose was introduced into Micromonospora rosaria IFO13697 cells, which produce the 16-membered macrolide antibiotic rosamicin containing a branched lactone and D-desosamine at the C-5 position. Although the M. rosaria TPMA0001 transconjugant exhibited low rosamicin productivity, two new compounds, IZI and IZII, were detected in the ethylacetate extract from the culture broth. IZI was identified as a mycinosyl rosamicin derivative, 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (MW 741), which has previously been synthesized by a bioconversion technique. This is the first report on production of mycinosyl rosamicin-derivatives by a engineered biosynthesis approach. The integration site PhiC31attB was identified on M. rosaria IFO13697 chromosome, and the site lay within an ORF coding a pirin homolog protein. The pSETmycinose could be useful for stimulating the production of "unnatural" natural mycinosyl compounds by various actinomycete strains using the bacteriophage PhiC31 att/int system.

Transcription factor activating enhancer-binding protein-2beta. A negative regulator of adiponectin gene expression.

We previously reported the association between the activating enhancer-binding protein-2beta (AP-2beta) transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with the disease-susceptible allele of AP-2beta showed stronger expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2beta leads to lipid accumulation by enhancing glucose transport and inducing insulin resistance in 3T3-L1 adipocytes. In this study we demonstrated that overexpression of AP-2beta in 3T3-L1 adipocytes decreased the expression and secretion of adiponectin and increased those of interleukin-6 (IL-6). Interestingly, the effects of AP-2beta on the expressions of adiponectin and IL-6 and the mechanisms by which AP-2beta modulated their expressions were different. We found that the promoter activity of adiponectin gene was inhibited by AP-2beta overexpression and enhanced by knockdown of endogenous AP-2beta, whereas IL-6 was unaffected. Electrophoretic mobility shift assays revealed the existence of putative responsive elements for AP-2beta and NF-YA in human and mouse adiponectin promoter regions, and mutation of this AP-2beta binding site abolished the inhibitory effect of AP-2beta. Furthermore, chromatin immunoprecipitation assays demonstrated that AP-2beta and NF-YA competitively bind to the same region of the adiponectin promoter. Our results clearly demonstrated that AP-2beta directly inhibits adiponectin gene expression by displacing NF-YA and binding to its promoter. We conclude that AP-2beta might modulate the expression of adiponectin by directly inhibiting its transcriptional activity.

Exploration of genes that encode a carbocycle-forming enzyme involved in biosynthesis of aminoglycoside antibiotics from the environmental DNA.

2-Deoxy-scyllo-inosose (DOI) synthase is the enzyme participating in biosynthesis of 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics. The gene which encodes the enzyme can be a marker for screening of DOS-containing aminoglycoside-producer and exploration of its biosynthetic gene. Further, this enzyme is expected to be of use in industry, because it converts sugar into 6-membered carbocycle. In the present study, we identified 21 clones encoding DOI synthase from environmental DNA by degenerate PCR. They were clearly divided into two groups. One appeared to derive from actinomycetes, and the other from non-actinomycetes. The latter group was larger (17 clones) than the former (four clones) despite the fact that only one strain of non-actinomycete was identified for DOS-containing aminoglycoside production. This result indicates that there are still many unidentified non-actinomycetes for DOS-containing aminoglycoside biosynthesis. We showed the possibility of identification of novel aminoglycoside-producing non-actinomycete from soil, and for development of more efficient enzymes for industrial use.

Intronic polymorphisms within TFAP2B regulate transcriptional activity and affect adipocytokine gene expression in differentiated adipocytes.

We have identified a gene encoding transcription factor activating enhancer binding protein-2beta (TFAP2B) as a candidate for conferring susceptibility to type 2 diabetes. Although we have also found that TFAP2B was preferentially expressed in adipose cells in a differentiation-dependent manner, the mechanisms by which the gene and gene polymorphisms contribute to conferring susceptibility to the disease have not yet been elucidated. The aim of this study was to evaluate the impact of the polymorphisms within the TFAP2B gene on conferring susceptibility to type 2 diabetes. We identified that a 300-bp DNA fragment in intron 1 of TFAP2B had significant enhancer activity, and the variations of this region affected this enhancer activity in differentiated adipocytes. In an experiment using adenovirus vectors encoding TFAP2B, the expression of TNF-alpha gene was shown to be elevated in the TFAP2B overexpressing cells compared with those in control cells. Furthermore, we demonstrated that the expression of TFAP2B was increased in the adipose tissues of subjects with the disease-susceptibility allele, and the plasma levels of TNF-alpha and high sensitivity C-reactive peptide were significantly elevated in the patients with the disease-susceptibility allele. These results suggest that TFAP2B may contribute to the pathogenesis of type 2 diabetes through regulation of adipocytokine gene expression, and that TFAP2B may be a promising target for treatment or prevention of this disease.

The transcription factor AP-2beta causes cell enlargement and insulin resistance in 3T3-L1 adipocytes.

We have reported the association of variations in the activating protein-2beta (AP-2beta) transcription factor gene with type 2 diabetes. This gene was preferentially expressed in 3T3-L1 adipocytes in a differentiation stage-dependent manner, and preliminary experiments showed that subjects with the disease-susceptible allele showed stronger expression in adipose tissue than those without the susceptible allele. Thus, we overexpressed the AP-2beta gene in 3T3-L1 adipocytes to clarify whether AP-2beta might play a crucial role in the pathogenesis of type 2 diabetes through dysregulation of adipocyte function. In cells overexpressing AP-2beta, cells increased in size by accumulation of triglycerides accompanied by enhanced glucose uptake. On the contrary, suppression of AP-2beta expression by small interfering RNA inhibited glucose uptake. Enhancement of glucose uptake by AP-2beta overexpression was attenuated by inhibitors of phospholipase C (PLC) and atypical protein kinase Czeta/lambda (PKCzeta/lambda), but not by a phosphatidylinositol 3-kinase (PI3-K) inhibitor. Consistently, we found activation of PLC and atypical PKC, but not PI3-K, by AP-2beta expression. Furthermore, overexpression of PLCgamma enhanced glucose uptake, and this activation was inhibited by an atypical PKC inhibitor, suggesting that the enhanced glucose uptake may be mediated through PLC and atypical PKCzeta/lambda, but not PI3-K. Moreover, we observed the increased tyrosine phosphorylation of Grb2-associated binder-1 (Gab1) and its association with PLCgamma, indicating that Gab1 may be involved in AP-2beta-induced PLCgamma activation. Finally, AP-2beta overexpression was found to relate to the impaired insulin signaling. We propose that AP-2beta is a candidate gene for producing adipocyte hypertrophy and may relate to the abnormal characteristics of adipocytes observed in obesity.

Genetic variations in the gene encoding TFAP2B are associated with type 2 diabetes mellitus.

To search a gene(s) conferring susceptibility to type 2 diabetes mellitus, we genotyped nearly 60,000 gene-based SNPs for Japanese patients and found evidence that the gene at chromosome 6p12 encoding transcription-factor-activating protein 2beta (TFAP2B) was a likely candidate in view of significant association of polymorphism in this gene with type 2 diabetes. Extensive analysis of this region identified that several variations within TFAP2B were significantly associated with type 2 diabetes [a variable number of tandem repeat locus: chi(2)=10.9, P=0.0009; odds ratio=1.57, 95% CI 1.20-2.06, intron 1+774 (G/T); chi(2)=11.6, P=0.0006; odds ratio=1.60, 95% CI 1.22-2.09, intron 1+2093 (A/C); chi(2)=12.2, P=0.0004; odds ratio=1.61, 95% CI 1.23-2.11]. The association of TFAP2B with type 2 diabetes was also observed in the UK population. These results suggest that TFAP2B might be a new candidate for conferring susceptibility to type 2 diabetes and contribute to the pathogenesis of type 2 diabetes.

Wnt5b partially inhibits canonical Wnt/beta-catenin signaling pathway and promotes adipogenesis in 3T3-L1 preadipocytes.

To elucidate the functional roles of Wnt5b in adipogenesis, we characterized gene expression profiles in Wnt5b overexpressing 3T3-L1 cells using microarray analysis. Of the approximately 20,000 genes screened, we found that 85 genes were up-regulated and 211 genes were down-regulated in 3T3-L1 cells overexpressing Wnt5b. Among the genes regulated by Wnt5b, the expressions of insulin like growth factor-1 (IGF-1), vascular endothelial growth factor-C (VEGF-C), and WNT1 inducible signaling pathway protein 1 (WISP-1), which were known to be up-regulated by Wnt1/beta-catenin signaling, were decreased in the Wnt5b overexpressing cells. This result was subsequently confirmed by real-time quantitative RT-PCR (IGF-1; 0.74+/-0.08 and 0.56+/-0.08, WISP-1; 0.71+/-0.03 and 0.56+/-0.08, and VEGF-C; 0.67+/-0.01 and 0.80+/-0.07, mean+/-SEM, compared with the control at zero and two days after induction of differentiation, respectively). We also found that Wnt5b overexpression in 3T3-L1 preadipocytes was able to partially prevent the inhibitory effect of Wnt3a on adipogenesis. Furthermore, the overexpression of Wnt5b was able to inhibit Wnt3a-induced activation of the canonical Wnt/beta-catenin pathway as evidenced by the reduced translocation of beta-catenin into the nucleus. These findings indicate that Wnt5b may promote adipogenesis in 3T3-L1 cells, at least in part, by antagonizing the canonical Wnt/beta-catenin pathway.

PKCepsilon induces interleukin-6 expression through the MAPK pathway in 3T3-L1 adipocytes.

Recent reports have suggested that PKCepsilon contributes to systemic insulin resistance, and is involved in the pathogenesis of type 2 diabetes, however, the exact mechanism is still unknown. To elucidate the possible involvement of PKCepsilon in the pathogenesis of type 2 diabetes, we examined the role of PKCepsilon in differentiated adipocytes using mouse 3T3-L1 adipocytes. We found that the over-expression of PKCepsilon resulted in the increase of IL-6 expression in differentiated adipocytes. This PKCepsilon-induced IL-6 expression could be completely inhibited by U0126, an inhibitor of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase. We also demonstrated that PKCepsilon increased the transcriptional activity of Est-like transcription factor (Elk-1) as well as the DNA-binding activity of activator protein-1 (AP-1) in differentiated 3T3-L1 adipocytes. These results suggest that PKCepsilon is able to increase IL-6 expression via the ERK-AP-1 pathway in differentiated adipocytes, and that PKCepsilon is involved in systemic insulin resistance by regulating plasma IL-6 concentrations.

Inhibition of experimental intimal thickening in mice lacking a novel G-protein-coupled receptor.

BACKGROUND: Vascular restenosis attributable to intimal thickening remains a major problem after percutaneous transluminal coronary angioplasty (PTCA). METHODS AND RESULTS: Through differential-display analysis, we have identified a novel gene whose expression was increased after catheter injury of rabbit aorta. The gene that is expressed predominantly in vascular smooth muscle cells encodes a novel protein with 7 transmembrane domains, and we termed it ITR (intimal thickness-related receptor). The ITR sequence contains a motif common to the Rhodopsin-like GPCR (G-protein-coupled receptor) superfamily. In vivo analyses of this gene revealed that expression of ITR protein increased with intimal thickening induced by cuff placement around murine femoral artery. Furthermore, ITR-knockout mice were found to be resistant to this experimental intimal thickening. CONCLUSIONS: ITR thus seems to be a novel receptor that may play a role in vascular remodeling and that may represent a good target for development of drugs in the prevention of vascular restenosis.