SM22α (Smooth Muscle Protein 22-α) Promoter-Driven IGF1R (Insulin-Like Growth Factor 1 Receptor) Deficiency Promotes Atherosclerosis.

Objective- IGF-1 (insulin-like growth factor 1) is a major autocrine/paracrine growth factor, which promotes cell proliferation, migration, and survival. We have shown previously that IGF-1 reduced atherosclerosis and promoted features of stable atherosclerotic plaque in Apoe-/- mice-an animal model of atherosclerosis. The aim of this study was to assess effects of smooth muscle cell (SMC) IGF-1 signaling on the atherosclerotic plaque. Approach and Results- We generated Apoe-/- mice with IGF1R (IGF-1 receptor) deficiency in SMC and fibroblasts (SM22α [smooth muscle protein 22 α]-CreKI/IGF1R-flox mice). IGF1R was decreased in the aorta and adventitia of SM22α-CreKI/IGF1R-flox mice and also in aortic SMC, embryonic, skin, and lung fibroblasts isolated from SM22α-CreKI/IGF1R-flox mice. IGF1R deficiency downregulated collagen mRNA-binding protein LARP6 (La ribonucleoprotein domain family, member 6) and vascular collagen, and mice exhibited growth retardation. The high-fat diet-fed SM22α-CreKI/IGF1R-flox mice had increased atherosclerotic burden and inflammatory responses. α-SMA (α-smooth muscle actin)-positive plaque cells had reduced proliferation and elevated apoptosis. SMC/fibroblast-targeted decline in IGF-1 signaling decreased atherosclerotic plaque SMC, markedly depleted collagen, reduced plaque fibrous cap, and increased plaque necrotic cores. Aortic SMC isolated from SM22α-CreKI/IGF1R-flox mice had decreased cell proliferation, migration, increased sensitivity to apoptosis, and these effects were associated with disruption of IGF-1-induced Akt signaling. Conclusions- IGF-1 signaling in SMC and in fibroblast is a critical determinant of normal vascular wall development and atheroprotection.

Roles of chondroitin sulfate proteoglycan 4 in fibrogenic/adipogenic differentiation in skeletal muscle tissues.

Intramuscular adipose tissue and fibrous tissue are observed in some skeletal muscle pathologies such as Duchenne muscular dystrophy and sarcopenia, and affect muscle strength and myogenesis. They originate from common fibrogenic/adipogenic cells in the skeletal muscle. Thus, elucidating the regulatory mechanisms underlying fibrogenic/adipogenic cell differentiation is an important step toward the mediation of these disorders. Previously, we established a highly adipogenic progenitor clone, 2G11, from rat skeletal muscle and showed that basic fibroblast growth factor (bFGF) is pro-adipogenic in these cells. Here, we demonstrated that 2G11 cells give rise to fibroblasts upon transforming growth factor (TGF)-ß1 stimulation, indicating that they possess mesenchymal progenitor cells (MPC)-like characteristics. The previously reported MPC marker PDGFRα is expressed in other cell populations. Accordingly, we produced monoclonal antibodies that specifically bind to 2G11 cell surface antigens and identified chondroitin sulfate proteoglycan 4 (CSPG4) as a potential MPC marker. Based on an RNA interference analysis, we found that CSPG4 is involved in both the pro-adipogenic effect of bFGF and in TGF-ß-induced alpha smooth muscle actin expression and stress fiber formation. By establishing an additional marker for MPC detection and characterizing its role in fibrogenic/adipogenic differentiation, these results will facilitate the development of effective treatments for skeletal muscle pathologies.

Insulin receptor substrates form high-molecular-mass complexes that modulate their availability to insulin/insulin-like growth factor-I receptor tyrosine kinases.

Insulin receptor substrates (IRSs) are phosphorylated by activated insulin/insulin-like growth factor (IGF)-I receptor tyrosine kinases. Phosphotyrosyl IRSs are recognized by signaling molecules possessing src homology region 2 (SH2) domains, which mediate various insulin/IGF bioactivities. However, we have shown that IRSs are also associated with other proteins by a phosphotyrosine-independent mechanism. Here, we demonstrated that IRSs form high-molecular-mass complexes (we named these complexes IRSomes) with various proteins and we elucidated their possible roles. Blue native-polyacrylamide gel electrophoresis of cell lysates revealed IRSome formation. Some proteins associated with IRSs in IRS-isoform-, cell-type-, or stimulus-specific manners. Results of the in vitro tyrosine phosphorylation assay indicated that tyrosine phosphorylation of IRS-1 by insulin receptor was decreased when IRS-1 was contained in IRSomes prepared from 3T3-L1 adipocytes treated with TNF-α. Also, tyrosine phosphorylation of IRS-2 by IGF-I receptor was increased when IRS-2 was contained in IRSomes prepared from FRTL-5 thyrocytes treated with dibutyryl cAMP. These results demonstrated that cytokine/hormone-induced formation of IRSomes modulates availability of IRSs to receptor tyrosine kinases.

Rbfox2 mediates exon 11 inclusion in insulin receptor pre-mRNA splicing in hepatoma cells.

Insulin receptor (IR) pre-mRNA undergoes alternative splicing that produces two isoforms, IR-A and IR-B. The ratio of IR-A to IR-B varies among tissues, which strongly suggests that IR mRNA alternative splicing is regulated in a tissue-specific manner. However, the precise molecular mechanism for IR alternative splicing remains to be elucidated, especially in liver. In this study, we have analyzed IR alternative splicing mechanism by preparing a mini-gene splicing reporter with rat genomic DNA. The splicing reporter that contains exon 11 and its flanking intronic sequences could reproduce alternative splicing pattern in rat hepatoma H4IIE cells. Introducing several deletions in introns of the reporter revealed that intron 11 contains the region near exon 11 essential to promote exon 11 inclusion. This region contains an UGCAUG sequence, a specific binding site for the Rbfox splicing regulator, and mutation in this sequence results in exon 11 skipping. Furthermore, RbFox2 knockdown in H4IIE cells enhanced exon 11 skipping of endogenous IR pre-mRNA. Lastly mutations in the SRSF3 binding site of exon11 together with the Rbfox2 binding site completely abolished exon 11 inclusion with a mini-gene reporter pre-mRNA. Our results indicate that RbFox2 and SRSF3 proteins mediate exon 11 inclusion in rat hepatoma cells.

Expression of Toll-like receptors (TLRs) and TLR4 response to lipopolysaccharide in hen oviduct.

The aim of this study was to determine the types of Toll-like receptors (TLRs) expressed in the hen oviduct, and to confirm that vaginal tissue expressing TLR4 responds to lipopolysaccharide (LPS). Healthy laying hens were intravenously or intravaginally injected with LPS, PBS or untreated. The expression pattern of TLRs in the whole oviduct and the effects of LPS on TLR4 and IL-1beta in the vagina were examined by semi-quantitative RT-PCR. The population of cells containing TLR4 protein was examined by immunohistochemistry. The expression of 6 types of TLRs (TLR1 type 2, TLR2, TLR3, TLR4, TLR5 and TLR7) were identified in all segments of the oviduct. The densities of PCR products for each TLR showed a tendency to be greater in the vagina than in the magnum and isthmus. Immunoreactive TLR4 was localized in the epithelial cells and leukocytes in the isthmus, uterus and vagina. Intravenous injection with LPS increased the TLR4 expression and the population of TLR4-immunopositive cells in the vagina. Intravaginal injection with LPS resulted in the increase of leukocytes in the mucosal tissues in association with an increase of TLR4 expression and TLR4-immunopositive cells in the vagina. The IL-1beta expression was also enhanced in a similar manner to that of TLR4. These results suggest that the hen oviduct expresses at least 6 types of TLRs including TLR4 with a greater expression in the vagina. Vaginal tissue expressing TLR4 responds to LPS and in turn upregulate cellular functions to synthesize cytokines. Such expression and functions of TLRs may play an essential role in oviductal innate immunity for host defense.

The Novel Functions of High-Molecular-Mass Complexes Containing Insulin Receptor Substrates in Mediation and Modulation of Insulin-Like Activities: Emerging Concept of Diverse Functions by IRS-Associated Proteins.

Insulin-like peptides, such as insulin-like growth factors (IGFs) and insulin, induce a variety of bioactivities, such as growth, differentiation, survival, increased anabolism, and decreased catabolism in many cell types and in vivo. In general, IGFs or insulin bind to IGF-I receptor (IGF-IR) or insulin receptor (IR), activating the receptor tyrosine kinase. Insulin receptor substrates (IRSs) are known to be major substrates of receptor kinases, mediating IGF/insulin signals to direct bioactivities. Recently, we discovered that IRSs form high-molecular-mass complexes (referred to here as IRSomes) even without IGF/insulin stimulation. These complexes contain proteins (referred to here as IRSAPs; IRS-associated proteins), which modulate tyrosine phosphorylation of IRSs by receptor kinases, control IRS stability, and determine intracellular localization of IRSs. In addition, in these complexes, we found not only proteins that are involved in RNA metabolism but also RNAs themselves. Thus, IRSAPs possibly contribute to modulation of IGF/insulin bioactivities. Since it is established that disorder of modulation of insulin-like activities causes various age-related diseases including cancer, we could propose that the IRSome is an important target for treatment of these diseases.

Insulin receptor substrate-1 associates with small nucleolar RNA which contributes to ribosome biogenesis.

Insulin receptor substrates (IRSs) are well known to play crucial roles in mediating intracellular signals of insulin-like growth factors (IGFs)/insulin. Previously, we showed that IRS-1 forms high molecular mass complexes containing RNAs. To identify RNAs in IRS-1 complexes, we performed ultraviolet (UV) cross-linking and immunoprecipitation analysis using HEK293 cells expressing FLAG-IRS-1 and FLAG-IRS-2. We detected the radioactive signals in the immunoprecipitates of FLAG-IRS-1 proportional to the UV irradiation, but not in the immunoprecipitates of FLAG-IRS-2, suggesting the direct contact of RNAs with IRS-1. RNAs cross-linked to IRS-1 were then amplified by RT-PCR, followed by sequence analysis. We isolated sequence tags attributed to 25 messenger RNAs and 8 non-coding RNAs, including small nucleolar RNAs (snoRNAs). We focused on the interaction of IRS-1 with U96A snoRNA (U96A) and its host Rack1 (receptor for activated C kinase 1) pre-mRNA. We confirmed the interaction of IRS-1 with U96A, and with RACK1 pre-mRNA by immunoprecipitation with IRS-1 followed by Northern blotting or RT-PCR analyses. Mature U96A in IRS-1(-/-) mouse embryonic fibroblasts was quantitatively less than WT. We also found that a part of nuclear IRS-1 is localized in the Cajal body, a nuclear subcompartment where snoRNA mature. The unanticipated function of IRS-1 in snoRNA biogenesis highlights the potential of RNA-associated IRS-1 complex to open a new line of investigation to dissect the novel mechanisms regulating IGFs/insulin-mediated biological events.

Insulin receptor substrate-1 (IRS-1) forms a ribonucleoprotein complex associated with polysomes.

Insulin receptor substrates (IRSs) are known to play important roles in mediating intracellular insulin-like growth factors (IGFs)/insulin signaling. In this study, we identified components of messenger ribonucleoprotein (mRNP) as IRS-1-associated proteins. IRS-1 complex formation analysis revealed that IRS-1 is incorporated into the complexes of molecular mass more than 1000 kDa, which were disrupted by treatment with RNase. Furthermore, oligo(dT) beads precipitated IRS-1 from cell lysates, showing that the IRS-1 complexes contained messenger RNA. Taken together with the data that IRS-1 was fractionated into the polysome-containing high-density fractions, we concluded that IRS-1 forms the novel complexes with mRNPs.