Role of Telokin in Regulating Murine Gastric Fundus Smooth Muscle Tension

Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from telokin(-/-) mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/W-V mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that telokin S13 is basally phosphorylated and that 8-BromocGMP and SNP increased basal telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased telokin phosphorylation and reduced MLC phosphorylation. In telokin(-/-) gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted telokin(-/-) gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8Bromo- cGMP, SNP, and nitrergic neurotransmission. However, basal telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased telokin Ser13 phosphorylation in both wild-type and W/W-V gastric fundus smooth muscles. Our findings indicate that telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to augment gastric fundus mechanical responses to inhibitory neurotransmission

STAT6 Deficiency Attenuates Myeloid Fibroblast Activation and Macrophage Polarization in Experimental Folic Acid Nephropathy

Renal fibrosis is a pathologic feature of chronic kidney disease, which can lead to end-stage kidney disease. Myeloid fibroblasts play a central role in the pathogenesis of renal fibrosis. However, the molecular mechanisms pertaining to myeloid fibroblast activation remain to be elucidated. In the present study, we examine the role of signal transducer and activator of transcription 6 (STAT6) in myeloid fibroblast activation, macrophage polarization, and renal fibrosis development in a mouse model of folic acid nephropathy. STAT6 is activated in the kidney with folic acid nephropathy. Compared with folic-acid-treated wild-type mice, STAT6 knockout mice had markedly reduced myeloid fibroblasts and myofibroblasts in the kidney with folic acid nephropathy. Furthermore, STAT6 knockout mice exhibited significantly less CD206 and PDGFR-β dual-positive fibroblast accumulation and M2 macrophage polarization in the kidney with folic acid nephropathy. Consistent with these findings, STAT6 knockout mice produced less extracellular matrix protein, exhibited less severe interstitial fibrosis, and preserved kidney function in folic acid nephropathy. Taken together, these results have shown that STAT6 plays a critical role in myeloid fibroblasts activation, M2 macrophage polarization, extracellular matrix protein production, and renal fibrosis development in folic acid nephropathy. Therefore, targeting STAT6 may provide a novel therapeutic strategy for fibrotic kidney disease

SNP dose-dependently increases telokin S13 phosphorylation in non-pre-contracted gastric fundus smooth muscles.

<p>A. Representative recording of the relaxation responses and representative western blot of phosphorylated and non-phosphorylated MLC evoked by 10Hz EFS, 0.1μM, 1μM, or 10μM SNP (n = 4). B. Average ratios ± SD of peak relaxation amplitudes evoked by 10Hz EFS, 0.1μM, 1μM, or 10μM SNP (*<i>P</i><0.01, ^#<i>P</i><0.05, n = 4). C. Average ratios ± SD and representative western blots of pS13:telokin evoked by 0.1μM, 1μM, or 10μM SNP (^#<i>P</i><0.05 compared to 0.1μM SNP or 1μM SNP, n = 4).</p

The relaxation responses of pre-contracted telokin^-/- gastric fundus smooth muscles are reduced compared to wild-type controls.

<p>Representative recordings of the relaxation responses of wild-type and telokin^-/- gastric fundus smooth muscles to (A) 10μM SNP, (B) 50μM 8Br-cGMP, and (C) 30 sec of 5Hz or 10Hz EFS. Representative recordings of the relaxation responses of wild-type and telokin^-/- gastric fundus smooth muscles evoked by 10μM SNP in the presence of (D) 1μM CCh or (E) 30mM KCl. F. Representative recording of the relaxation responses evoked by 30 sec of 5Hz or 10Hz EFS in wild-type and telokin^-/- gastric fundus smooth muscles incubated with 30mM KCl and 1μM atropine, 1μM phentolamine, and 1μM propranolol.</p

Telokin phosphorylation in non-contracted gastric fundus smooth muscles is not increased by nitrergic neurotransmission.

<p>A. Representative recording of the relaxation responses evoked by 30 sec of 5Hz or 10Hz EFS of muscles incubated in 1μM atropine, 1μM phentolamine, and 1μM propranolol. Representative western blots of the phosphorylation of telokin S13 (B), CPI-17 T38 (C), MYPT1 T853 and T696 (D), and MLC S19 (E) evoked by 30 sec of 5Hz or 10Hz EFS of muscles incubated in 1μM atropine, 1μM phentolamine, and 1μM propranolol. F. Average ratios ± SD of pS13:telokin, pT38:CPI-17, pT696:MYPT1, pT853:MYPT1, and pS19:MLC evoked by 30 sec of 5Hz or 10Hz EFS of muscles incubated in 1μM atropine, 1μM phentolamine, and 1μM propranolol (<i>P</i>>0.05, n = 6).</p

Telokin phosphorylation is increased by incubation of non-contracted gastric fundus smooth muscles with compounds that elevate cGMP.

<p>A. Representative recordings of the relaxation responses evoked by 10μM SNP in the absence or presence of 1μM ODQ (n = 6, each), or 50μM 8Br-cGMP (n = 6). B. Representative western blots of telokin S13 phosphorylation in untreated control muscles and muscles incubated with 50μM 8Br-cGMP for 5 min (n = 6) or 10μM SNP (n = 6) for 1 min. C. Average ratios ± SD of pS13:telokin in the absence or presence of 50μM 8Br-cGMP (n = 6) or 10μM SNP. *<i>P</i><0.01 compared to untreated controls.</p

The contractile responses of telokin^-/- gastric fundus smooth muscles are increased compared to wild-type controls.

<p>A. Representative recordings of the contractile responses of wild-type and telokin^-/- gastric fundus smooth muscles to 1μM CCh (n = 4). B. Representative recordings of the contractile responses of wild-type and telokin^-/- gastric fundus smooth muscles to 15mM, 30mM, and 60mM KCl. C. Average integrals and peak amplitudes of contraction ± SD of wild-type and telokin^-/- gastric fundus smooth muscles to 15mM, 30mM, and 60mM KCl (^#<i>P</i><0.05 compared to wild-type, n = 8). D. Representative recordings of the contractile responses of wild-type and telokin^-/- gastric fundus smooth muscles incubated with 100μM LNNA and 1μM MRS2500 to 30 sec of 1Hz, 5Hz, 10Hz, or 20Hz EFS. E. Average integrals and peak amplitudes of contraction ± SD of wild-type and telokin^-/- gastric fundus smooth muscles incubated with 100μM LNNA and 1μM MRS2500 to 30 sec of 1Hz, 5Hz, 10Hz, or 20Hz EFS KCl (^#<i>P</i><0.05 compared to wild-type, n = 4). F. Representative western blots of phosphorylated and non-phosphorylated MLC in wild-type and telokin^-/- gastric fundus smooth muscles.</p

Telokin S13 phosphorylation is not increased by nitrergic neurotransmission in <i>W/W</i>^<i>V</i> gastric fundus smooth muscles.

<p>Representative recordings of the relaxation responses evoked by 30 sec of 5Hz EFS and 10Hz EFS (A), or 10μM SNP (B) from wild-type and <i>W/W</i>^<i>V</i> fundus muscles. B. Representative western blot of phosphorylated and non-phosphorylated MLC from wild-type and <i>W/W</i>^<i>V</i> fundus muscles treated with 10μM SNP. C. Average ratios ± SD of pS13:telokin and representative western blots of phosphorylated telokin S13 and telokin from wild-type and <i>W/W</i>^<i>V</i> muscles stimulated by 10Hz/30 sec EFS, or incubated with 10μM SNP. *<i>P</i><0.01 compared to untreated wild-type or <i>W/W</i>^<i>V</i> fundus muscle controls, n = 4.</p

Telokin phosphorylation in gastric fundus smooth muscles pre-contracted with 1μM CCh is increased by compounds that elevate cGMP.

<p>A. Representative recordings of the contractile response to 1μM CCh, and the relaxation responses evoked by 50μM 8Br-cGMP (n = 6) or 10μM SNP (n = 6) in the presence of 1μM CCh. Average ratios ± SD of pS13:telokin, pT38:CPI-17, pT696: MYPT1, pT853:MYPT1, and pS19:MLC and representative western blots of the phosphorylation of (B) telokin S13, (C) CPI-17 T38, (D) MYPT1 T696 and T853, and (E) MLC S19 evoked by 50μM 8Br-cGMP (n = 6) or 10μM SNP (n = 6) in the presence of 30mM KCl. *<i>P</i><0.01, ^#<i>P</i><0.05, compared to untreated controls. ^‡<i>P</i><0.05 compared to 1μM CCh treated muscles.</p