Calcium and the replication of human vascular smooth muscle cells: studies on the activation and translocation of extracellular signal regulated kinase (ERK) and cyclin D1 expression.
Since the precise role of sarcoplasmic reticular Ca2+ in mediating vascular smooth muscle cells (VSMC) proliferation is unknown, the effect of pre-incubation with thapsigargin on extracellular signal regulated kinase (ERK) activation, the translocation of activated of ERK 1/2 to the nucleus, cyclin D1 expression, the onset of S phase and cytosolic Ca2+ levels were studied. Human saphenous vein VSMCs (hVSMC) were incubated with 10 nM thapsigargin for 24 h followed by stimulation with fetal calf serum and the activation of ERK1/2 and cyclin D1 assessed by western blotting, the intracellular distribution of ERK1/2 using indirect immunofluorescence, the onset of S-phase with the incorporation of bromodeoxyuridine and sarcoplasmic reticular Ca2+ status using FURA-2. Thapsigargin had a marginal effect on ERK1/2 activation only at 5 min and 10 min after stimulation with fetal calf serum. In contrast, the rapid translocation of ERK1/2 to the nucleus was completely blocked by thapsigargin. S phase was delayed by 8 h by thapsigargin which co-incided with the recovery of cytosolic Ca2+ levels and cyclin D1 expression. It is concluded that the inhibitory effect of thapsigargin (depletion of Ca2+ pools) on hVSMC replication is mediated through the inhibition of translocation of activated ERK1/2 to the nucleus and not to the phosphorylation of ERK, per se, which in turn prevents cyclin D1 expression and thus progression of the cell cycle.
Shukla
Nilima
N
Bristol Heart Institute, Department of Cardiac Surgery, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, UK.
Rowe
Daniel
D
Hinton
Jane
J
Angelini
Gianni D
GD
Jeremy
Jamie Y
JY
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002467:Cell Nucleus / Q000187:drug effects / Q000502:physiology; D019938:Cyclin D1 / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000473:pathology / Q000502:physiology*; D016196:S Phase / Q000187:drug effects / Q000502:physiology; D012501:Saphenous Vein / Q000187:drug effects / Q000473:pathology / Q000502:physiology; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014178:Translocation, Genetic
D019938:Cyclin D1; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D012710:Serum Albumin, Bovine; D000001:Calcimycin; D004533:Egtazic Acid; D015759:Ionomycin; D019284:Thapsigargin; D048049:Extracellular Signal-Regulated MAP Kinases; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium