Tomohiro Manabe

Involvement of gp130-mediated signaling in pressure overload-induced activation of the JAK/STAT pathway in rodent heart

SummaryPreviously, we showed that the JAK/STAT pathway was activated in pressure-overloaded rat heart, and that angiotensin II was partially involved in this activation. The present study was designed to investigate whether gp130-mediated signaling is involved in this activation, and if so, which interleukin (IL)-6 family cytokine is involved. Pressure overload was produced by ligation of the abdominal aorta of Wistar rats or ICR mice. IP-Western blot was performed to detect tyrosine phosphorylation of STATs, gp130, and the association of gp130 with JAK kinases. The serum concentration of IL-6 was measured by enzyme-linked immunosorbent assay. Expression of IL-6, IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), and cardiotrophin-1 (CT-1) mRNA was quantitated. After pressure overload, rapid phosphorylation of STAT1 and STAT3 was observed at 5min, STAT1 was rephosphorylated at 60min, and intense phosphorylation of STAT3 was observed at 60min. Both the phosphorylation of gp130 and the association of gp130 with JAK1 and JAK2 were increased after pressure overload. IL-6 was significantly increased by two-fold in the pressure-overloaded rats. Only CT-1 mRNA expression could be detected by Northern blot, and it increased after pressure overload. Reverse transcription-polymerase chain reaction revealed that IL-6 mRNA expression was increased 9.5-fold. IL-11, LIF, CNTF, and OSM expression were unaffected by pressure overload. These results suggested that gp130mediated signaling was involved in the pressure overload-induced activation of the JAK/STAT pathway, and that IL-6 and CT-1 might be involved in this activation.

Identification of a novel gene, LDOC1, down-regulated in cancer cell lines

By screening for differentially expressed genes in cancer cells, using the RNA differential display (DD) technique, we identified a novel cDNA, LDOC1, that is down-regulated in some cancer cell lines. A Northern blot analysis revealed no expression in pancreatic and gastric cancer cell lines but ubiquitous expression in normal human tissues. This new gene was mapped on chromosome Xq27 and the predicted protein sequence showed no similarity to known sequences in the database except for a leucine zipper-like motif at the N-terminal region and a proline-rich region that shares marked similarity to an SH3-binding domain. In an enhanced green fluorescent protein (EGFP) assay, the EGFP-LDOC1 fusion protein was localized in the nucleus. Although the function of LDOC1 is still unknown, our results suggest that this novel gene codes for a nuclear protein, and down-regulation of LDOC1 may have an important role in the development and/or progression of some cancers.

Characterization of Insulin-Like Growth Factor-1–Induced Activation of the JAK/STAT Pathway in Rat Cardiomyocytes

This study was designed to investigate whether insulin-like growth factor-1 (IGF-1) transduces signaling through the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in cardiomyocytes and to assess the upstream signals of serine and tyrosine phosphorylation of STAT family proteins. Primary cultured neonatal rat cardiomyocytes were stimulated with IGF-1 (10(-8) mol/L). JAK1, but not JAK2 or Tyk2, was phosphorylated by IGF-1 as early as 2 minutes and peaked at 5 minutes. IGF-1 induced both tyrosine and serine phosphorylation of STAT1 and STAT3. Tyrosine phosphorylation of STAT1 peaked at 15 minutes and correlated with that of JAK1, whereas that of STAT3 was sustained up to 120 minutes and was dissociated from the activation of JAK1. Tyrosine phosphorylation of STAT3 was unaffected by the preincubation with CV11974 (AT(1) blocker), TAK044 (endothelin-1 receptor blocker), RX435 (anti-gp130 blocking antibody), PD98058, wortmannin, EDTA, or KN62 but was significantly attenuated by BAPTA-AM and chelerythrine. The time course of a gel mobility shift of SIE (sis-inducing element) coincided with the phosphorylation of STAT3. Serine phosphorylation of STAT1 peaked at 30 minutes and that of STAT3 was observed from 5 to 60 minutes. These results indicated that (1) IGF-1 activated JAK1 but not JAK2 or Tyk2 in rat cardiomyocytes; (2) IGF-1 induced both tyrosine and serine phosphorylation of STAT1 and STAT3; and (3) the tyrosine phosphorylation of STAT3 was not caused by JAK1 alone, and protein kinase C and intracellular Ca(2+) were required for phosphorylation.

Identification of a novel gene, DAM1, amplified at chromosome 1p13.3-21 region in human breast cancer cell lines.

Screening for differentially expressed genes in cancer cell lines by the RNA differential display (DD) technique identified a novel mRNA that encodes a 26-kDa protein and is up-regulated in MCF-7 and BT-20 breast cancer cells. The predicted amino acid sequence showed 38% homology to Caenorhabditis elegans T12A2.7 protein, indicating that this is a possible human homologue for C. elegans T12A2.7 protein. The mRNA, designated DAM1 (DNA amplified in mammary carcinoma), was mapped at the 1p13.3-21 region, which is frequently altered in human breast cancer. By Southern blot analysis, we confirmed that the up-regulation of this novel gene is associated with gene amplification in MCF-7 (10-fold) and BT-20 (5-fold) cells. Our findings suggest that the DAM1 gene is a novel gene up-regulated by amplification in human breast cancer cell lines.

Hypertrophic stimuli augment expression of cMG1/ERF-1, a putative zinc-finger motif transcription factor, in rat cardiomyocytes.

We isolated the gene for cMG1/ERF‐1, a known putative zinc‐finger transcription factor, by differential display of mRNA extracted from cardiomyocytes with and without leukemia inhibitory factor (LIF) stimulation. LIF induced cMG1/ERF‐1 mRNA at 15 min, and levels peaked at 10‐fold initial levels at 30 min. cMG1/ERF‐1 expression was inhibited by AG490 (JAK2 inhibitor) and genistein, but was unaffected by PD98059 or wortmannin. Phenylephrine, angiotensin II and endothelin‐1 also induced cMG1/ERF‐1 expression. Mechanical stretch in vitro and acute pressure overload in vivo increased cMG1/ERF‐1 expression. To our knowledge, this is the first report showing that the cMG1/ERF‐1 gene can be induced by various hypertrophic stimuli, and that Janus kinase 2 is involved in this process.

NEW STRATEGY OF MEDICAL THERAPY TO LEFT VENTRICULAR DYSFUNCTION WITHOUT SIGNIFICANT CORONARY STENOSIS BY USING ACETYLCHOLINE PROVOCATION

Results: In either patient with positive ACh-test (n=13) or with negative ACh-test (n=19), LV function was severely depressed (LVEF: 32 vs. 37%, ns; BNP: 903 vs. 580; ns). In all of patients with positive ACh-test, ACh-CAG showed multi-vessel diffuse coronary spasm with remarkable ECG changes. Among positive ACh-test group, all of three LV biopsy specimens were negative, and only one of 10 cases (10%) showed an abnormal MRI delayed enhancement (DE). On the other hand, among the negative ACh-test group, LV biopsy in 2 of 3 cases and MRI-DE findings in 5 of 10 cases showed more frequently abnormal findings compatible with cardiomyopathy than among the positive ACh-test group (60%, vs. 10%, p<0.05). Among the positive ACh-test group, depressed LV function improved after starting Ca antagonist during 3 months (LVEF: 32±12% to 49±13%, p<0.01; BNP: 903±565 to 80±40, p<0.01), however, among the negative ACh-test group, depressed LV improved under treatment of antagonist during 8 months (LVEF: 37±11% to 44±11%, p<0.01; BNP: 580±470 to 144±149, p<0.01).