RESUMEN
Maladaptive bladder muscle overgrowth and de-differentiation in human bladder obstructive conditions is instigated by coordinate responses to three stimuli: mechanical strain, tissue hypoxia, and extracellular matrix remodeling.( 1,2) Pathway analysis of genes induced by obstructive models of injury in bladder smooth muscle cells (BSMCs) identified a mammalian target of rapamycin (mTOR)-specific inhibitor as a potential pharmacological inhibitor. Strain-induced mTOR-specific S6K activation segregated differently from ERK1/2 activation in intact bladder ex vivo. Though rapamycin's antiproliferative effects in vascular smooth muscle cells are well known, its effects on BSMCs were previously unknown. Rapamycin significantly inhibited proliferation of BSMCs in response to mechanical strain, hypoxia, and denatured collagen. Rapamycin inhibited S6K at mTOR-sensitive phosphorylation sites in response to strain and hypoxia. Rapamycin also supported smooth muscle actin expression in response to strain or hypoxia-induced de-differentiation. Importantly, strain plus hypoxia synergistically augmented mTOR-dependent S6K activation, Mmp7 expression and proliferation. Forced expression of wild-type and constitutively active S6K resulted in loss of smooth muscle actin expression. Decreased smooth muscle actin, increased Mmp7 levels and mTOR pathway activation during in vivo partial bladder obstruction paralleled our in vitro studies. These results point to a coordinate role for mTOR in BSMCs responses to the three stimuli and a potential new therapeutic target for myopathic bladder disease.
Asunto(s)
Desdiferenciación Celular , Matriz Extracelular/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Estrés Mecánico , Vejiga Urinaria/patología , Actinas/metabolismo , Animales , Bovinos , Desdiferenciación Celular/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Metaloproteinasa 7 de la Matriz/metabolismo , Mitógenos/farmacología , Modelos Biológicos , Miocitos del Músculo Liso/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Vejiga Urinaria/efectos de los fármacosRESUMEN
Excessive stretch of the bladder can lead to wall thickening including the growth of bladder smooth muscle cells (BSMC). Only three phospho-proteins (JNK, p38, and PI3K) have been previously shown to participate in stretch-induced BSMC growth. CD1 mouse bladders were hyper- or non-distended by our ex vivo bladder distention model and screened, by a commercial screening method, for phosphorylated signaling proteins. This uncovered a factor previously unexamined for its role in bladder stretch injury: signal transducer and activator of transcription 3 (STAT3). STAT3 was assessed for its role in mitogen- and stretch-induced BSMC proliferation. Proliferation was assessed by 3H-thymidine incorporation/cell counting in response to mitogenic stimulation or to stretch on silastic collagen or carboxyl-coated membranes. JAK2, upstream of STAT3, was inhibited by AG490 (2 microM). Ex vivo distention of bladders activated a discrete number of kinases, including two MAPK pathways (JNK and ERK2) and STAT3. STAT3 signaling was activated during hyperdistention of intact bladder and by stretch and mitogenic treatments of BSMC in vitro. JAK2/STAT3 inhibition by AG490 blocked mitogen- and stretch-induced BSMC proliferation. Thus, BSMC stretch responses may involve the recruitment of both growth factor and mechanically induced BSMC growth responses integrated by a common signaling pathway, STAT3.