Glutamine switches vascular smooth muscle cells to synthetic phenotype through inhibiting miR-143 expression and upregulating THY1 expression.

AIMS: Vascular smooth muscle cells (VSMCs) are involved in the pathogenesis of many human cardiovascular diseases. They modulate their phenotype from "contractile" to "synthetic" in response to changes in local environmental cues. How glutamine regulates the differentiation of VSMCs and the underlying mechanisms remain largely unknown. MAIN METHODS: Here, we explored the effects of various doses of glutamine (0 mM, 1 mM, 2 mM, and 4 mM) on the proliferation, migration, and phenotypic switch of human VSMCs in vitro. Glutamine dose-dependently enhanced VSMC proliferation, and markedly increased VSMC migration. KEY FINDINGS: Notably, glutamine promoted the phenotypic switch of VSMCs towards a synthetic phenotype, as evidenced by significantly decreased expression of contractile markers myosin heavy chain 11 (MYH11) and calponin while increased expression of synthetic markers collagen I and vimentin. Importantly, these changes upon glutamine treatments were attenuated after additional treatments with glutamine metabolism inhibitor BPTES. Additionally, glutamine downregulated miR-143 expression, and miR-143 inactivation alone resulted in enhanced proliferation, migration, and promoted the synthetic phenotype of VSMCs. Moreover, Thy-1 cell surface antigen (THY1) was validated as a downstream target of miR-143, and THY1 expression was upregulated by glutamine in VSMCs. Furthermore, either miR-143 overexpression or THY1 silencing abolished the effect of glutamine on proliferation, migration, and phenotypic switch of VSMCs, supporting a novel glutamine-miR-143-THY1 pathway in modulating VSMC functions. SIGNIFICANCE: This study demonstrated a novel mechanism of glutamine in modulation of VSMC phenotypic switch by targeting miR-143 and THY1, and provides significant insight on targeted therapy of patients with cardiovascular diseases.

[Expression and purification of truncated fragment of extracellular segment of sodium pump alpha3 subunit in Escherichia coli by in-fusion technology].

OBJECTIVE: To construct prokaryotic expression system for expressing, purifying and identifying truncated fragment of extra-cellular segment of sodium pump alpha3 subunit with pGEX-6P-1 GST gene fusion system in Escherichia coli by in-fusion technology. METHODS: According to the conservative sequence of M1-M2 and M3-M4 extra-cellular gene fragments of sodium pump a3 subunit, which published in GenBank, a serial of primers and gene fragments was designed, and directly synthesized to fuse the above two gene fragments. The fusion gene was fused with gene-specific primers by PCR, and then fusion gene fragment was fused into the single stranded homology regions of vector pGEX-6P-1 by in-fusion cloning to construct recombinant vector pGEX-Trf-alpha3 (Truncated fragment of extracellular segment of sodium pump alpha3 subunit, Trf-alpha3). After DH10bac was transferred with it, the pGEX-Trf-alpha3 plasmid was purified and identified by PCR and sequenced. Then the recombinant plasmid pGEX-Trf-alpha3 was expressed in E. coli BL21 cells, inducted by IPTG. GST-Trf-alpha3 fusion protein was purified with Glutathione Sepharose 4B purifying system and analyzed by SDS-PAGE. RESULTS: The results of PCR and sequencing demonstrated that the M1-M2 and M3-M4 extra-cellular gene was inserted in plasmid pGEX-6P-1 vector successfully. And the sequence was correct. Protein sequence analysis showed that the GST-Trf-alpha3 fusion protein was consisted of 262 amino-acid residues. Relative molecular mass in theory was 33.22 X 10(3). The amount of recombinant protein was 10% of the total bacteria protein. The soluble fusion protein was about 80.8%. After affinity purification, the purity of GST-Trf-alpha3 fusion protein was over 95%. There was some extent binding activity between GST-Trf-alpha3 fusion protein and ouabain, but the activity was very low. CONCLUSION: Prokaryotic expression system for expressing truncated fragment of extra-cellular segment of sodium pump alpha3 subunit with pGEX-6P-1 GST gene fusion system in Escherichia coli by in-fusion technology had been constructed. The purified method had also established. High purified GST-Trf-alpha3 fusion protein was obtained. These have found the foundation of further study on its biological function and potential pharmacology function.

[Activity identification of peptide containing rat sodium pump α2 subunit M1-M2 extramembrane fragment in vitro].

In order to explore the activity of a peptide containing rat sodium pump α2 subunit M1-M2 extramembrane fragment (RES2 derivative) in vitro, the peptide (Leu-Ala-Ala-Met-Glu-Asp-Glu-Pro-Ser-Asn-Asp-Asn-Gly-Gly-Gly-Ser) was synthesized by peptide synthesizer with Fmoc method and purified by high performance liquid chromatography (HPLC). Its binding activity was identified by radioligand-receptor binding assay (RRA) and its bioactivity was measured by erythrocyte (86)Rb uptake. The results of saturation binding experiment and competitive binding experiment showed that the synthesized RES2 derivative had the capability to bind to (3)H-ouabain. The dissociation constant (K(d)) was 38.46 nmol/L and IC(50) was 6.353 nmol/L. Erythrocyte (86)Rb uptake experiment showed that the RES2 derivative blocked the inhibitory effect of ouabain on the sodium pump on erythrocyte membrane in a dose-dependent manner. The results showed that the RES2 derivative is capable of binding to ouabain and improving the activity of sodium pump on erythrocyte membrane, suggesting that the RES2 derivative might become an effective antihypertensive drug in the future.