Smad proteins differentially regulate obesity-induced glucose and lipid abnormalities and inflammation via class-specific control of AMPK-related kinase MPK38/MELK activity

Smad proteins have been implicated in metabolic processes, but little is known about how they regulate metabolism. Because Smad 2, 3, 4, and 7 have previously been shown to interact with murine protein serine–threonine kinase 38 (MPK38), an AMP‐activated protein kinase (AMPK)-related kinase that has been implicated in obesity-associated metabolic defects, we investigated whether Smad proteins regulate metabolic processes via MPK38. Smads2/3/4 increased, but Smad7 decreased, MPK38-mediated apoptosis signal-regulating kinase-1 (ASK1)/transforming growth factor-β (TGF-β)/p53 signaling. However, MPK38-mediated phosphorylation-defective Smad mutants (Smad2 S245A, Smad3 S204A, Smad4 S343A, and Smad7 T96A) had no such effect. In addition, Smads2/3/4 increased, but Smad7 decreased, the stability of MPK38. Consistent with this, Smads2/3/4 attenuated complex formation between MPK38 and its negative regulator thioredoxin (Trx), whereas Smad7 increased this complex formation. However, an opposite effect was observed on complex formation between MPK38 and its positive regulator zinc-finger-like protein 9 (ZPR9). When Smads were overexpressed in high-fat diet (HFD)-fed obese mice using an adenoviral delivery system, Smads2/3/4 improved, but Smad7 worsened, obesity-associated metabolic parameters and inflammation in a MPK38 phosphorylation-dependent manner. These findings suggest that Smad proteins have class-specific impacts on obesity-associated metabolism by differentially regulating MPK38 activity in diet-induced obese mice.

To assess the effects of Smad2/4 on TGF-β-mediated apoptosis, HaCaT cells were transfected with various concentrations of vectors encoding WT or mutant Smads2/4 (0.5 and 1 μg) and/or WT and K40R MPK38 (0.4 μg), as indicated, together with an expression vector encoding GFP (1 μg). After treatment of the transfected cells with TGF-β1 (2 ng/ml, 20 h), GFP-based apoptotic cell death was determined (right panels). ***p < 0.001 compared with MPK38 alone in the presence of TGF-β1. c Differential regulation of MPK38-dependent p53- a Size distribution analysis of adipocytes was performed in HFD-fed mice infected with the indicated adenoviruses, as described previously 8 . n = 6 per group. b In vitro 3 H-2-deoxyglucose uptake by soleus muscles was measured in the presence or absence of human insulin (10 mU/ml) (left panels). n = 6 per group, *p < 0.05, **p < 0.01, ***p < 0.001 compared with control treated with insulin, determined by two-way ANOVA. IRS-PI3K signaling was evaluated by immunoblot analyses (right panels) after in vivo insulin stimulation by injection into the inferior vena cava (n = 2 per group). c Serum level of glucose, determined using an automated serum analyzer. n = 6 per group, *p < 0.05, **p < 0.01 compared with control.
ASK1/TGF-β/p53 signaling and the kinase activity of MPK38 in hepatocytes and adipocytes derived from HFD-fed mice infected with the indicated adenoviruses were analyzed by immunoblot analysis using the indicated antibodies and the anti-phospho-specific antibodies described in Fig. 1a Obese mice display lower Smads2/3/4 expression and higher levels of Smad7 expression compared to control lean mice, leading to the downregulation of ASK1/TGF-β/p53 signaling through differential regulation of MPK38 kinase activity. Supplementary Fig. 6 Comparison of the expression levels of Smads2/3/4/7 and the activation levels of p53 signaling between mice fed a control chow diet and a HFD.
Primary hepatocytes derived from Chow-and HFD-fed C57BL/6 mice (a) and isolated 5 hepatocytes treated (+) or untreated (-) with 5FU (0.38 mM, 30 h) (b) were subjected to immunoblot analysis using the indicated antibodies to examine the protein expression levels.
The experiments were repeated at least three times with similar results. Supplementary Fig. 7 Demonstration of in vivo phosphorylation of Smads2/3/4/7 by MPK38 in CRISPR/Cas9 Smad knock-in cells.
a Immunoprecipitated MPK38 (upper panels) and recombinant MPK38 (lower panels) were assayed for their kinase activity in the presence of Smad2/3/4/7 immunoprecipitates obtained from cell lysates of WT and clonal CRISPR/Cas9 Smad knock-in isolates as substrates. b In vitro kinase assays were carried out with immunoprecipitated Smad2/3/4/7 as substrates in the absence of MPK38. Each number represents an individual clone number of CRISPR/Cas9 Smad KI isolates. WT, wild-type; Cons., control; KI, knock-in.