Enhancement of leptin receptor signaling by SOCS3 deficiency induces development of gastric tumors in mice.

Leptin acts on its receptor (ObR) in the hypothalamus to inhibit food intake and energy expenditure. Leptin and ObR are also expressed in the gastrointestinal tract; however, the physiological significance of leptin signaling in the gut remains uncertain. Suppressor of cytokine signaling 3 (SOCS3) is a key negative feedback regulator of ObR-mediated signaling in the hypothalamus. We now show that gastrointestinal epithelial cell-specific SOCS3 conditional knockout (T3b-SOCS3 cKO) mice developed gastric tumors by enhancing leptin production and the ObRb/signal transducer and activator of transcription 3 (STAT3) signaling pathway. All T3b-SOCS3 cKO mice developed tumors in the stomach but not in the bowels by 2 months of age, even though the SOCS3 deletion occurred in both the epithelium of stomach and bowels. The tumors developed in the absence of the inflammatory response and all cKO mice died within 6 months. These tumors displayed pathology and molecular alterations, such as an increase in MUC2 (Mucin 2, oligomeric mucus/gel-forming) and TFF3 (trefoil factor 3), resembling human intestinal-type gastric tumors. Administration of antileptin antibody to T3b-SOCS3 cKO mice reduced hyperplasia of gastric mucosa, which is the step of the initiation of gastric tumor. These data suggest that SOCS3 is an antigastric tumor gene that suppresses leptin overexpression and ObRb/STAT3 hyperactivation, supporting the hypothesis that the leptin/ObRb/STAT3 axis accelerates tumorigenesis and that it may represent a new therapeutic target for the treatment of gastric cancer.

Green tea catechin inhibits lipopolysaccharide-induced bone resorption in vivo.

BACKGROUND AND OBJECTIVE: Bone resorption is positively regulated by receptor activator of nuclear factor-kappaB ligand (RANKL). Pro-inflammatory cytokines, such as interleukin (IL)-1beta, promote RANKL expression by stromal cells and osteoblasts. Green tea catechin (GTC) has beneficial effects on human health and has been reported to inhibit osteoclast formation in an in vitro co-culture system. However, there has been no investigation of the effect of GTC on periodontal bone resorption in vivo. We therefore investigated whether GTC has an inhibitory effect on lipopolysaccharide (LPS)-induced bone resorption. MATERIAL AND METHODS: Escherichia coli (E. coli) LPS or LPS with GTC was injected a total of 10 times, once every 48 h, into the gingivae of BALB/c mice. Another group of mice, housed with free access to water containing GTC throughout the experimental period, were also injected with LPS in a similar manner. RESULTS: The alveolar bone resorption and IL-1beta expression induced by LPS in gingival tissue were significantly decreased by injection or oral administration of GTC. Furthermore, when GTC was added to the medium, decreased responses to LPS were observed in CD14-expressing Chinese hamster ovary (CHO) reporter cells, which express CD25 through LPS-induced nuclear factor-kappaB (NF-kappaB) activation. These findings demonstrated that GTC inhibits nuclear translocation of NF-kappaB activated by LPS. In addition, osteoclasts were generated from mouse bone marrow macrophages cultured in a medium containing RANKL and macrophage colony-stimulating factor with or without GTC. The number of osteoclasts was decreased in dose-dependent manner when GTC was added to the culture medium. CONCLUSION: These results suggest that GTC suppresses LPS-induced bone resorption by inhibiting IL-1beta production or by directly inhibiting osteoclastogenesis.

Cloning and characterization of a novel p21(Cip1/Waf1)-interacting zinc finger protein, ciz1.

p21(Cip1/Waf1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N- and C-terminal domains, respectively. Here, we report a novel p21(Cip1/Waf1)-interacting protein, Ciz1 (for Cip1 interacting zinc finger protein), which contains polyglutamine repeats and glutamine-rich region in the N-terminus as well as three zinc-finger motifs and one MH3 (matrin 3-homologous domain 3) in the C-terminal region. Ciz1 bound to the N-terminal, the CDK2-interacting part of p21(Cip1/Waf1), and the interaction was disrupted by the overexpression of CDK2. A region of about 150 amino acids containing the first zinc-finger motif in Ciz1 was the binding site for p21(Cip1/Waf1). When Ciz1 and p21(Cip1/Waf1) were individually overexpressed in U2-OS cells, they mostly localized in the nucleus. However, coexpression of Ciz1 induced cytoplasmic distribution of p21(Cip1/Waf1). These data indicate that Ciz1 is a unique nuclear protein that regulates the cellular localization of p21(Cip1/Waf1).

Cloning and expression of the Ca2+ channel alpha1C and beta2a subunits from guinea pig heart.

Complimentary DNA clones encoding the alpha1C and beta2a subunits of guinea-pig cardiac L-type Ca2+ channels were isolated using the PCR method. The open reading frame encoded 2,169 amino acids for the alpha1C and 597 amino acids for the beta2a subunit. The proteins showed 94.2 and 94.8%, respectively, identity to the respective subunit of the rabbit protein. The message size of the guinea pig alpha1C and beta2a subunits was 8.0 and 3.5/4.0 kb, respectively. RT-PCR analysis revealed that the alpha1C subunit is expressed exclusively in the heart, while the beta2a subunit is expressed in the heart, cerebellum, whole brain, and stomach. The alpha1C and beta2a subunits are transiently expressed in BHK (baby hamster kidney) cells, and the channel currents were studied using the whole-cell patch clamp technique in medium containing 30 mM Ba2+. In cells expressing alpha1C alone, the Ba2+ current was activated at -30 mV and more positive potentials and peaked at about 10 mV. The co-expression of beta2a with alpha1C did not affect the voltage-dependence of the current, but increased the peak current and accelerated current decay. In cells transfected with guinea pig alpha1C and rabbit beta1+alpha2/delta, a Ba2+ current comparable to those in native myocytes was observed. The Ba2+ current can be blocked completely by nifedipine and is enhanced 3-fold by Bay K 8644. On the other hand, neither forskolin nor okadaic acid affects the Ba2+ current, suggesting that cAMP-mediated modulation is not easily reproduced in transfected cells, unlike that seen in native cardiac myocytes.

Cloning and characterization of a novel class II phosphoinositide 3-kinase containing C2 domain.

Phosphoinositide 3-kinases (PI3Ks) have been shown to play critical roles in cell growth, differentiation, survival, and vesicular transport. Class II PI3Ks have been recently identified in mouse and human (PI3K-C2 alpha/m-p170/m-cpk and HsC2-PI3K) and in Drosophila (PI3K 68D/cpk) which contain C2 domain at the C-terminus. However, their physiological function is largely unknown. We report here cloning and characterization of murine PI3K-C2 gamma, a novel class II PI3K. The catalytic domain as well as C2 domain are highly conserved in the Class II PI3K family, while the N-terminal regions of these proteins share little similarity. Unlike other Class II PI3Ks, PI3K-C2 gamma exclusively expressed in the liver, and a N-terminal truncated form was found in lung and a certain hematopoietic cell line. Specific antiserum against PI3K-C2 gamma precipitated PI3K activity from the membrane fraction of mouse liver but not from heart. Recombinant PI3K-C2 gamma exhibited a restricted lipid substrate specificity; it phosphorylated phosphatidylinositol (PtdIns) and PtdIns4P but not PtdIns(4,5)P2. Deletion mutations revealed that both the N-terminal region and the C2 domain were critical for enzymatic activity. The murine PI3K-C2 gamma gene locus was mapped to the distal region of mouse chromosome 6 in a region of homology with human chromosome 12p, which is distinct from the position of HsC2-PI3K. Cloning and biochemical characterization of the third member of class II PI3Ks provide a new insight into the function of this subfamily of PI3Ks.

STAT5 activation correlates with erythropoietin receptor-mediated erythroid differentiation of an erythroleukemia cell line.

Interaction between erythropoietin (EPO) and its membrane receptor induces the proliferation and differentiation of erythroid progenitors. EPO has been shown to activate the JAK2-STAT5 pathway in various hematopoietic cell lines, although the physiological role of this pathway is unclear. We have previously shown that epidermal growth factor activates a chimeric receptor bearing the extracellular domain of the epidermal growth factor receptor linked to the cytoplasmic domain of the EPO receptor, resulting in proliferation of interleukin-3-dependent hematopoietic cells and erythroid differentiation (globin synthesis) of EPO-responsive erythroleukemia cells. In the present study, we introduced various deletion and tyrosine to phenylalanine substitution in the cytoplasmic domain of the chimeric receptor and expressed these mutant chimeras in an EPO-responsive erythroleukemia cell line, ELM-I-1. Mutant chimeric receptors retaining either Tyr343 or Tyr401 could activate STAT5, judged by tyrosine-phosphorylation of STAT5 and induction of CIS, a target gene of STAT5. These mutants were able to induce erythroid differentiation. However, a chimeric receptor containing both Y343F and Y401F mutations could not activate STAT5 nor induce erythroid differentiation. Thus, Tyr343 or Tyr401 of the EPO receptor are independently necessary for erythroid differentiation as well as STAT5 activation. Moreover, exogenous expression of dominant-negative STAT5 suppressed EPO-dependent erythroid differentiation. These findings suggest that STAT5 plays an important role in erythroid differentiation through the EPO receptor cytoplasmic domain.

Treatment of refractory hyperparathyroidism in patients on hemodialysis by intermittent oral administration of 1,25(OH)2vitamin D3.

Intermittent oral administration of high dose 1,25(OH)2vitamin D3 was conducted in 7 patients associated with treatment-resistant secondary hyperparathyroidism (2nd HPT) on hemodialysis (HD) therapy. Each patient had a long history of HD therapy (range: 101-181 months, 145 +/- 29 months). Although serum calcium levels were maintained under the upper limit of the normal range with the appropriate dose of 1 alpha(OH)vitamin D3 every day before the present therapy, 2nd HPT could not have been controlled. The dose of 2-3 micrograms of 1,25(OH)2vitamin D3 3 times a week could successfully suppress serum levels of parathyroid hormone (iPTH) in all 7 patients after 20-32 weeks. The vitamin was given in the evening before each HD session and the dose and frequency of administration were dependent of the serum calcium level in each patient. After 20 weeks the iPTH-C and iPTH-intact levels decreased significantly from 35.0 +/- 15.8 to 18.6 +/- 11.7 ng/ml and from 533.2 +/- 200.0 to 249.5 +/- 136.2 pg/ml, respectively. The frequency of harmful elevations of serum calcium levels was not significantly increased in comparison with that in the previous period of the study, because serum calcium levels were strictly monitored with frequent checks. In conclusion, we could safely obtain an effect similar to the intravenous administration of the vitamin through the intermittent administration of a high oral dose 1,25(OH)2vitamin D3 in the treatment of refractory 2nd HPT in patients on HD therapy.

[The effect of corticosteroid on lymphokine-activated killer (LAK) activity and the change of membrane antigen phenotype on PBMC after IL2 incubation with reference to the evaluation in vitro].

In the treatment of human cancer, the efficacy of the adoptive transfer of lymphokine-activated killer (LAK) cells combined with interleukin 2 (IL2) has been emphasized. However, administration of large doses of IL2 may cause significant adverse effects, such as "capillary leak syndrome". Moreover, the administration of corticosteroid may reduce the toxicity of IL2. However the efficacy of adoptive immunotherapy is abrogated by the administration of corticosteroid. In this context, the effect of corticosteroid on LAK activity and the change of membrane antigen phenotype on PBMC (peripheral blood mononuclear cells) was studied in vitro by using methylprednisolone (M-PSL). LAK cells were induced from PBMC, which had been separated from the peripheral blood of normal donors, and incubated with IL2 for 4 days. LAK activity was measured in a 4 h chromium release assay against K-562 cell and Daudi cell. The suppression of LAK activity by M-PSL depended on both concentration and working period of M-PSL. This inhibition was recognized even at rather low densities, such as 0.5 nmol/ml M-PSL. And the proliferation of PBMC after IL2 incubation, which was measured by incorporation of [3H]TdR, was reduced dose-dependently by M-PSL. In contrast, pretreatment within 24 hours of PBMC with M-PSL resulted in no effect on LAK activity. Furthermore M-PSL had no effect when added directly to a 4 h chromium release assay. The analysis of membrane antigen phenotype on PBMC was performed by the direct immunofluorescence method using FITC-labeled monoclonal antibodies. The proportions of T cells and NK cells, which were justified as precursors of LAK cells, increased after IL2 incubation both with and without M-PSL. Therefore no effect of M-PSL was confirmed in those circumstances. The ratio of HLA-DR (+) cells increased after IL2 incubation without M-PSL, while M-PSL reduced expression of this antigen. In contrast, IL2 receptor (CD25)(+) cells, markers of T cell and NK cell activation, significantly increased after IL2 incubation with or without M-PSL. These results suggest that the inhibitory effect of M-PSL on LAK activation was caused not by preventing a triggering process of activation of precursor cells, but by a possible inhibition of proliferation, though other effects of corticosteroid remain to be elucidated. Also, it is emphasized that caution should be exercised in the administration of corticosteroid in adoptive immunotherapy because of the inhibitory effect induced by M-PSL.