Selective Tuberous Sclerosis Complex 1 Gene Deletion in Smooth Muscle Activates Mammalian Target of Rapamycin Signaling and Induces Pulmonary Hypertension

Am J Respir Cell Mol Biol. 2016 Sep;55(3):352-67. doi: 10.1165/rcmb.2015-0339OC.

Abstract

Constitutive activation of the mammalian target of rapamycin (mTOR) complexes mTORC1 and mTORC2 is associated with pulmonary hypertension (PH) and sustained growth of pulmonary artery (PA) smooth muscle cells (SMCs). We investigated whether selective mTORC1 activation in SMCs induced by deleting the negative mTORC1 regulator tuberous sclerosis complex 1 gene (TSC1) was sufficient to produce PH in mice. Mice expressing Cre recombinase under SM22 promoter control were crossed with TSC1(LoxP/LoxP) mice to generate SM22-TSC1(-/-) mice. At 8 weeks of age, SM22-TSC1(-/-) mice exhibited PH with marked increases in distal PA muscularization and Ki67-positive PASMC counts, without systemic hypertension or cardiac dysfunction. Marked activation of the mTORC1 substrates S6 kinase and 4E-BP and the mTORC2 substrates p-Akt(Ser473) and glycogen synthase kinase 3 was found in the lungs and pulmonary vessels of SM22-TSC1(-/-) mice when compared with control mice. Treatment with 5 mg/kg rapamycin for 3 weeks to inhibit mTORC1 and mTORC2 fully reversed PH in SM22-TSC1(-/-) mice. In chronically hypoxic mice and SM22-5HTT(+) mice exhibiting PH associated with mTORC1 and mTORC2 activation, PH was maximally attenuated by low-dose rapamycin associated with selective mTORC1 inhibition. Cultured PASMCs from SM22-TSC1(-/-), SM22-5HTT(+), and chronically hypoxic mice exhibited similar sustained growth-rate enhancement and constitutive mTORC1 and mTORC2 activation; both effects were abolished by rapamycin. Deletion of the downstream mTORC1 effectors S6 kinase 1/2 in mice also activated mTOR signaling and induced PH. We concluded that activation of mTORC1 signaling leads to increased PASMC proliferation and subsequent PH development.

Keywords: pulmonary hypertension; rapamycin; smooth muscle.

MeSH terms

  • Animals
  • Cell Proliferation / drug effects
  • Cells, Cultured
  • Chronic Disease
  • Gene Deletion*
  • Hyperplasia
  • Hypertension, Pulmonary / diagnostic imaging
  • Hypertension, Pulmonary / metabolism*
  • Hypoxia / complications
  • Hypoxia / metabolism
  • Hypoxia / pathology
  • Lung / blood supply
  • Lung / pathology
  • Male
  • Metformin / pharmacology
  • Mice
  • Microfilament Proteins / metabolism
  • Muscle Proteins / metabolism
  • Muscle, Smooth / drug effects
  • Muscle, Smooth / metabolism*
  • Myocytes, Smooth Muscle / drug effects
  • Myocytes, Smooth Muscle / metabolism
  • Pulmonary Artery / pathology
  • Ribosomal Protein S6 Kinases, 90-kDa / metabolism
  • Signal Transduction* / drug effects
  • Sirolimus / pharmacology
  • TOR Serine-Threonine Kinases / metabolism*
  • Tuberous Sclerosis Complex 1 Protein
  • Tumor Suppressor Proteins / metabolism*

Substances

  • Microfilament Proteins
  • Muscle Proteins
  • Tagln protein, mouse
  • Tsc1 protein, mouse
  • Tuberous Sclerosis Complex 1 Protein
  • Tumor Suppressor Proteins
  • Metformin
  • Ribosomal Protein S6 Kinases, 90-kDa
  • TOR Serine-Threonine Kinases
  • ribosomal protein S6 kinase, 90kDa, polypeptide 3
  • Sirolimus