Leiomodin 2 (Lmod2) is an actin-binding protein that has been implicated in the regulation of striated muscle thin filament assembly; its physiological function has yet to be studied. We found that knockout of Lmod2 in mice results in abnormally short thin filaments in the heart. We also discovered that Lmod2 functions to elongate thin filaments by promoting actin assembly and dynamics at thin filament pointed ends. Lmod2-KO mice die as juveniles with hearts displaying contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy. Lmod2-null cardiomyocytes produce less contractile force than wild type when plated on micropillar arrays. Introduction of GFP-Lmod2 via adeno-associated viral transduction elongates thin filaments and rescues structural and functional defects observed in Lmod2-KO mice, extending their lifespan to adulthood. Thus, to our knowledge, Lmod2 is the first identified mammalian protein that functions to elongate actin filaments in the heart; it is essential for cardiac thin filaments to reach a mature length and is required for efficient contractile force and proper heart function during development.actin-thin filaments | cardiomyopathy | cytoskeletal dynamics S triated muscle cells contain arrays of protein filaments assembled into contractile units that are nearly crystalline in structure. Efficient contraction at the molecular level is predicated upon accurate overlap of actin-containing thin and myosin-containing thick filaments. Therefore, proper control of filament assembly is absolutely critical.In striated muscle it is currently thought that the thin-filament pointed end capping protein tropomodulin (Tmod) is the predominant regulator of thin filament length, with Tmod1 being the sole isoform expressed in cardiomyocytes (1). Extensive in vitro work has revealed that Tmod1 uses two actin-and two tropomyosin-binding sites to associate with the end of the thin filament and to prevent addition or loss of actin monomers, thereby controlling length of the thin filament (2-7). Tmod1 is essential for life; Tmod1-KO mice are embryonic lethal because of cardiac defects (8-11).Identification of additional but structurally different members of the Tmod family of proteins, the leiomodins (Lmods), raises the possibility that thin filament lengths are not regulated solely by Tmod at thin filament pointed ends (12). Although there are three Lmod genes (Lmod1-3), Lmod2 and 3 are expressed in striated muscle with Lmod2 being the predominant isoform in cardiac muscle and Lmod3 the predominant isoform in skeletal muscle (12-16). The Lmods share ∼40% sequence identity at the protein level with the Tmods but do not contain a recognizable second tropomyosin-binding domain and have an additional C-terminal extension that includes a proline-rich region and an actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domain (12, 17). Lmod2 has been proposed to be the long-sought muscle actin filament nucleator because it robustly nucleates actin filament formation in ...
The nuclear vitamin D receptor (VDR) mediates the actions of 1,25-dihydroxyvitamin D 3 (1,25D) to regulate gene transcription. Recently, the secondary bile acid, lithocholate, was recognized as a novel VDR ligand. Using reporter gene and mammalian two-hybrid systems, immunoblotting, competitive ligand displacement, and quantitative real time PCR, we identified curcumin (CM), a turmeric-derived bioactive polyphenol, as a likely additional novel ligand for VDR. CM (10 −5 M) activated transcription of a luciferase plasmid containing the distal vitamin D responsive element from the human CYP3A4 gene at levels comparable to 1,25D (10 −8 M) in transfected human colon cancer cells (Caco-2). While CM also activated transcription via a retinoid X receptor (RXR) responsive element, activation of the glucocorticoid receptor (GR) by CM was negligible. Competition binding assays with radiolabeled 1,25D confirmed that CM binds directly to VDR. In mammalian two hybrid assays employing transfected Caco-2 cells, CM (10 −5 M) increased the ability of VDR to recruit its heterodimeric partner, RXR, and steroid receptor coactivator-1 (SRC-1). Real time PCR studies revealed that CM-bound VDR can activate VDR target genes CYP3A4, CYP24, p21, and TRPV6 in Caco-2 cells. Numerous studies have shown chemoprotection by CM against intestinal cancers via a variety of mechanisms. Small intestine and colon are important VDRexpressing tissues where 1,25D has known anticancer properties that may, in part, be elicited by activation of CYP-mediated xenobiotic detoxification and/or up-regulation of the tumor suppressor p21. Our results suggest the novel hypothesis that nutritionally-derived CM facilitates chemoprevention via direct binding to, and activation of, VDR.
The nuclear vitamin D receptor (VDR) modulates gene transcription in 1,25-dihydroxyvitamin D3 (1,25D) target tissues such as kidney, intestine, and bone. VDR is also expressed in heart, and 1,25D deficiency may play a role in the acceleration of cardiovascular disease. Employing a yeast two-hybrid system and a human heart library, using both a 1,25D-independent and 1,25D-dependent screen, we discovered six candidate VDR interacting proteins (VIPs). These novel VIPs include CXXC5, FASTK, NR4A1, TPM2, MYL3 and XIRP1. Mammalian two-hybrid assays as well as GST pull-downs were used to confirm VIP-VDR interaction, and the combination of these two assays reveals that CXXC5, XIRP1, FASTK and NR4A1 interactions with VDR may be modulated by 1,25D. The functional effects of these VIPs on 1,25D-mediated gene expression were explored in transcriptional assays employing three separate and distinct 1,25D-responsive element (VDRE)-driven luciferase reporter genes in transfected Caco-2 and HEK-293 cells, and in a C2C12 myoblast line. FASTK and TPM2 activated expression in all cell line and promoter contexts, while CXXC5 and XIRP1 exhibited differing effects depending on the cell line and promoter employed, suggesting promoter and cell-specific effects of these unique VIPs on VDR signaling. Further evaluation of the interaction between CXXC5 and VDR revealed that CXXC5 acts in a dose-dependent manner to stimulate VDR-mediated transcription on select VDREs. Identification of novel heart VIPs and their influence on VDR activity may increase our understanding of how vitamin D impacts cardiac physiology and may facilitate development of VDR/VIP drug analogs to combat heart disease.
In the present study we report that the undernourished rats during fetal life submitted to a neonatal recovery regime had a return to normal metabolic and physical growth conditions during the nursing period, and that their food consumption was more than controls, from weaning until adult age. However, in spite of the metabolic and physical recovery of the gestational undernourished rats, the activity of brain tryptophan-5-hydroxylase (TrpOH) remained elevated accompanied by an increase in the concentration of the neurotransmitter, serotonin (5-HT). Besides, the current observation confirms and extends to previous results, that an increase in brain 5-HT content, in L-tryptophan (L-Trp) concentration and in the activity of TrpOH, in undernourished rats occurs not only during gestation and lactation periods, but it lasts until adulthood. The increase in the activity of TrpOH observed during the fetal stage and continuing to postnatal life in undernourished rats seems to be secondary to an increased transport of plasma L-Trp to their brain. These findings suggest the hypothesis that the mechanism of accelerated synthesis of brain 5-HT in the adult nutritionally recovered animals, may not depended on the increased availability of free plasma L-Trp observed in the undernourished rats, but might be due to a specific change in the TrpOH structure, supported by previous results showing different kinetic and phosphorylating properties. Our observations also suggest that the increased food intake in the recovered animals imply changes in feeding behavior possibly related to the altered serotonin brain neurotransmission.
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