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

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.

Smoothelin-like protein 1 promotes insulin sensitivity and modulates the contractile properties of endometrial epithelial cells with insulin resistance

“…SMTNL1 negatively influences the migration of endometrial epithelial cells; SMTNL1 overexpression decreases MP activity and increases the phosphorylation of MLC20 (Figures 2A, C, 3C), especially in the P4 and GDB models. This finding is in agreement with a previous study showing that SMTNL1 overexpression in C2C12 myoblasts hampers cell migration, possibly through inhibition of MP (16). Moreover, inhibition of MP also reduced cell migration in HepG2 cells (15).…”

“…SMTNL1 promoted glucose uptake in a progesteronedependent manner and increased glycogen content in Ishikawa cells, compensating for insulin resistance (Figures 7A, B). This phenomenon was previously described in insulin-resistant myotubules; SMTNL1-overexpression initiated glycolysis (14) and regulated hexokinase at the gene expression level (16).…”

“…The confocal microscopy protocol was previously described (16). Briefly, the actin cytoskeleton was stained with Texas-Red Phalloidin (yellow) and nuclei were labeled with DAPI (4′, 6diamidino-2-phenylindole) (blue).…”

“…Semi-quantitative Western blot analyses were conducted, as previously described (16) with modifications as follows. Protein (50 µg) was loaded onto 4-20% precast Criterion gel (Bio-Rad Laboratories) and separated at 200 V. Nitrocellulose membranes were blocked in 5% (w/v) bovine serum albumin/tris-buffered saline containing 0.1% (v/v) Tween 20 (BSA/TBST) for at least 1.5 hours.…”

“…In mouse and differentiated skeletal muscle cell culture models of insulin resistance, SMTNL1 can attenuate the effects of altered IRS-1 serine phosphorylation induced by the over-activation of Ser/Thr protein kinases (14,15). By reducing the expression of novel type PKCϵ (nPKCϵ), SMTNL1 can downregulate the activity of ERK 1/2 MAPK, resulting in decreased IRS-1 Ser612 phosphorylation, which was boosted by in vitro hyperinsulinemichyperglycemic conditions (14,16). Thus, SMTNL1 prevents the dissociation of the IRS-1-PI3K-Akt-mTOR pathway and acts as an insulin-sensitizing agent in our in vitro rodent model.…”