Insulin increases the expression of contractile phenotypic markers in airway smooth muscle.
We have previously demonstrated that long-term exposure of bovine tracheal smooth muscle (BTSM) strips to insulin induces a functional hypercontractile phenotype. To elucidate molecular mechanisms by which insulin might induce maturation of contractile phenotype airway smooth muscle (ASM) cells, we investigated effects of insulin stimulation in serum-free primary BTSM cell cultures on protein accumulation of specific contractile phenotypic markers and on the abundance and stability of mRNA encoding these markers. In addition, we used microscopy to assess insulin effects on ASM cell morphology, phenotype, and induction of phosphatidylinositol (PI) 3-kinase signaling. It was demonstrated that protein and mRNA levels of smooth muscle-specific contractile phenotypic markers, including sm-myosin, are significantly increased after stimulation of cultured BTSM cells with insulin (1 microM) for 8 days compared with cells treated with serum-free media, whereas mRNA stability was unaffected. In addition, insulin treatment promoted the formation of large, elongate ASM cells, characterized by dramatic accumulation of contractile phenotype marker proteins and phosphorylated p70(S6K) (downstream target of PI 3-kinase associated with ASM maturation). Insulin effects on protein accumulation and cell morphology were abrogated by combined pretreatment with the Rho kinase inhibitor Y-27632 (1 microM) or the PI 3-kinase inhibitor LY-294002 (10 microM), indicating that insulin increases the expression of contractile phenotypic markers in BTSM in a Rho kinase- and PI 3-kinase-dependent fashion. In conclusion, insulin increases transcription and protein expression of contractile phenotypic markers in ASM. This could have important implications for the use of recently approved aerosolized insulin formulations in diabetes mellitus.
Schaafsma
Dedmer
D
Dept. of Molecular Pharmacology, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. d.schaafsma@rug.nl
McNeill
Karol D
KD
Stelmack
Gerald L
GL
Gosens
Reinoud
R
Baarsma
Hoeke A
HA
Dekkers
Bart G J
BG
Frohwerk
Erin
E
Penninks
Jelte-Maarten
JM
Sharma
Pawan
P
Ens
Karen M
KM
Nelemans
S Adriaan
SA
Zaagsma
Johan
J
Halayko
Andrew J
AJ
Meurs
Herman
H
D000577:Amides / Q000494:pharmacology; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002417:Cattle; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000378:metabolism / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects* / Q000235:genetics; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D009924:Organ Culture Techniques; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D024745:Smooth Muscle Myosins / Q000378:metabolism; D013997:Time Factors; D014132:Trachea / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D054460:rho-Associated Kinases; D000096985:Calponins
D000577:Amides; D002135:Calcium-Binding Proteins; D002867:Chromones; D003285:Contractile Proteins; D007004:Hypoglycemic Agents; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011725:Pyridines; D012333:RNA, Messenger; D017526:Receptor, IGF Type 1; D017527:Receptor, IGF Type 2; D011972:Receptor, Insulin; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D024745:Smooth Muscle Myosins; D054460:rho-Associated Kinases; D000096985:Calponins; C108830:Y 27632; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one