Tuberous sclerosis complex (TSC) is a inheritable genetic disorder characterized by the development of hamartomas, which are unusual tumor-like growth found in a variety of tissues and organs. Two genes are implicated in TSC, TSC1 and TSC2, respectively encoding hamartin and tuberin. Tuberin and hamartin function together as a complex to inhibit target of rapamycin (TOR)-mediated signaling to S6 kinase and participate in the control of cell size. The hamartin/tuberin complex inhibits the insulin pathway downstream of Akt. From a surgically removed angiomyolipoma (AML) of a TSC2 patient we have isolated -actin-positive smooth muscle cells bearing mutation on exon 18, a substitution A/T (2110) leading to a stop codon, with loss of heterozigosity (LOH) for TSC2 gene. These cells were, then, named A+. S6K is constitutively activated as expected, while IGF-I is abundantly released and involved in cell survival rather than proliferation. The addition of EGF to the cell medium is necessary to promote proliferation of TSC2-/- smooth muscle cells. Antibodies to IGF-I or EGF receptors cause blockade in cell proliferation with a drastic decrease in cell number beginning 5 days after exposure, total cell loss is observed within 12 days. Full length wild-type human tuberin was successfully expressed after retroviral transduction of TSC2 gene in TSC2-/- smooth muscle cells and the rate of cell proliferation returned normal, the EGF-growth dependency was lost, thus it appears that EGF requirement for A+ cell growth is due to the lack of tuberin. TSC2-/- A+ cells are positively labeled by HMB45 antibody, this is a marker for TSC and LAM cells. Following TSC2 transfection,TSC2-/- A+ cells were negative for HMB45. It has been shown that in cells containing TSC2 mutations and in cells lacking tuberin from the Eker rat, p70S6K and its substrate S6 are hyperphosphorylated. In TSC2-transfected A+ cells the extent of Akt and S6 phosphorylation was reduced to normal levels, with the expression of Akt and S6 unaltered. Also the extent of PTEN phosphorylation was higher in TSC2-/- A+ cells, but this decreased drastically following the reintroduction of TSC2 gene. 2 hours IGF-1 (50ng/ml) exposure slightly activated PI3K in TSC2-/- A+ cells, this was, however, not affected by the classic inhibitors such as LY294002 (LY) and wortmannin. The sensitivity to these inhibitors was restored by the reintroduction of the TSC2 gene in TSC2-/- A+ cells. In this condition, LY (20 M) addition reduced both basal and IGF-1-induced Akt phosphorylation, and inhibited activation of S6K1 and PTEN. Thus the TSC2 retroviral transduction, restored the sensitivity of PTEN phosphorylation to IGF-1 exposure, and suggest a parallel activation and deactivation for Akt and PTEN in TSC. PD98059 (PD), a specific inhibitor of ERK, did not affect Akt, S6 and S6K1 phosphorylation either in transfected cells and TSC2-/- A+ cell. The phenotype of TSC2-/- A+ cells was also asseseed after antiEGFR and rapamycin exposure. When A+ cells were exposed at plating time to antiEGFR (5g/ml) the labelling with HMB45 was down-regulated within 5 days. A smaller effect was observed with rapamycin (5ng/ml). Rapamycin blocked the excessive proliferation rate of TSC2-/- A+ cell when added at plating time, but a delay of 3 hours caused the complete loss of effect. AntiEGFR was effective when added in both conditions. In conclusion our study shows that lack of tuberin affects greatly the proliferation and IGF-I related metabolism in smooth muscle cells, the EGF dependency for growth and the insensitivity to IGF-I pathway inhibitors is reversed by the reintroduction of the TSC2 gene. The superior effects of antiEGFR versus rapamycin suggest a novel therapeutic strategy for TSC.
Reversion of the phenotype of human TSC2-/- smooth muscle cells / E. Lesma, V. Grande, E. Isaia, A.M. Di Giulio, A. Gorio. ((Intervento presentato al convegno International Research Symphosium Tuberous Sclerosis Complex tenutosi a Roma nel 2007.
|Titolo:||Reversion of the phenotype of human TSC2-/- smooth muscle cells|
LESMA, ELENA ANNA (Primo)
GRANDE, VERA (Secondo)
DI GIULIO, ANNA MARIA (Penultimo)
GORIO, ALFREDO (Ultimo)
|Data di pubblicazione:||24-mag-2007|
|Parole Chiave:||TSC2-/- ; smooth muscle cells|
|Settore Scientifico Disciplinare:||Settore BIO/14 - Farmacologia|
|Citazione:||Reversion of the phenotype of human TSC2-/- smooth muscle cells / E. Lesma, V. Grande, E. Isaia, A.M. Di Giulio, A. Gorio. ((Intervento presentato al convegno International Research Symphosium Tuberous Sclerosis Complex tenutosi a Roma nel 2007.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|