Smoothelin-Like Protein 1 Regulates the Thyroid Hormone-Induced Homeostasis and Remodeling of C2C12 Cells via the Modulation of Myosin Phosphatase.

Evelin Major,Ilka Keller,Ferenc Erdődi,Dániel Horváth,István Tamás,Beáta Lontay

doi:10.3390/ijms221910293

Evelin Major, Ilka Keller + Show 4 more

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https://doi.org/10.3390/ijms221910293

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Abstract

The pathological elevation of the active thyroid hormone (T3) level results in the manifestation of hyperthyroidism, which is associated with alterations in the differentiation and contractile function of skeletal muscle (SKM). Myosin phosphatase (MP) is a major cellular regulator that hydrolyzes the phosphoserine of phosphorylated myosin II light chain. MP consists of an MYPT1/2 regulatory and a protein phosphatase 1 catalytic subunit. Smoothelin-like protein 1 (SMTNL1) is known to inhibit MP by directly binding to MP as well as by suppressing the expression of MYPT1 at the transcriptional level. Supraphysiological vs. physiological concentration of T3 were applied on C2C12 myoblasts and differentiated myotubes in combination with the overexpression of SMTNL1 to assess the role and regulation of MP under these conditions. In non-differentiated myoblasts, MP included MYPT1 in the holoenzyme complex and its expression and activity was regulated by SMTNL1, affecting the phosphorylation level of MLC20 assessed using semi-quantitative Western blot analysis. SMTNL1 negatively influenced the migration and cytoskeletal remodeling of myoblasts measured by high content screening. In contrast, in myotubes, the expression of MYPT2 but not MYPT1 increased in a T3-dependent and SMTNL1-independent manner. T3 treatment combined with SMTNL1 overexpression impeded the activity of MP. In addition, MP interacted with Na+/K+-ATPase and dephosphorylated its inhibitory phosphorylation sites, identifying this protein as a novel MP substrate. These findings may help us gain a better understanding of myopathy, muscle weakness and the disorder of muscle regeneration in hyperthyroid patients.

Highlights

Our results demonstrate that desmin levels were significantly elevated in the NT-FT-Smoothelin-like protein 1 (SMTNL1)-transfected cells compared to the empty vector-transfected control on Day three (Figure 1A)
We examined the expression of a late myogenic marker, myosin heavy chain (MyHC), the expression of which increases sharply during regular differentiation [30]
NT-FT-SMTNL1 overexpressing cells showed stronger fluorescent signals even at Day zero of differentiation, which was more pronounced on Day five compared to the control. These results demonstrate that SMTNL1 overexpression leads to the upregulation of early and late myogenic markers, suggesting that SMTNL1 promotes myoblast differentiation

Summary

Introduction

Thyroid hormones (THs) have important regulatory actions in skeletal muscle (SKM) [1,2,3]. The main forms of thyroid hormone produced and secreted by the thyroid gland are tetraiodothyronine (T4 ) and 3,30 ,5-triiodothyronine (T3 ). Intracellular monodeiodination of T4 by deiodinase type II (DIO2) produces T3 , the active form of thyroid hormone in SKM [5]. The final level of thyroid hormone signaling control is given by the expression of nuclear thyroid hormone receptors (TRs), which positively or negatively regulate the transcription of TH responsive genes [3]. Functional TRs mediate the vast majority of TH actions on the level of gene transcription; so-called non-genomic effects using other target molecules have been reported as well [6]

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