Activation of the cell membrane angiotensin AT2 receptors in human leiomyosarcoma cells induces differentiation and apoptosis by a PPARγ - dependent mechanism

Angiotensin II (Ang II), the main effector peptide of the renin-angiotensin system (RAS), acting on AT(1) and AT(2) receptors participates in the regulation of proliferation, differentiation and apoptosis in tumour cells. The peroxisome-proliferator activated receptor gamma (PPAR gamma) and its ligands exert anti-tumour effects in various human cancer cell lines. The present study investigates the effects initiated by AT(1)- and AT(2) receptor stimulation in SK-UT-1 cells, a human leiomyosarcoma cell line, and clarifies the role of the PPAR gamma in the AT(2) receptor-induced differentiation and apoptosis. Selective stimulation of AT(1)- and AT(2) receptors was achieved by incubation of the cells with Ang II (10(-6) M) in the presence of the selective AT(2) receptor antagonist, PD 123177 (10(-6) M) and the AT(1) receptor antagonist, losartan (10(-5) M), respectively, the selective PPAR gamma antagonist, GW 9662, was used at concentration 10(-6) M. The expression of smooth muscle cell differentiation markers, SM22 alpha and calponin, was analysed at RNA- and protein levels using RT PCR and Western blot, which was also used to quantify Bcl-2-, Bax- and cleaved caspase-3 proteins. The translocation of the AT(2)-receptor interacting protein 1 (ATIP1) to the nuclei was studied by Western blot and immunofluorescence staining. The mitochondrial status and the metabolic activity in response to AT(1)- and AT(2) receptor activation were assessed by the quantification of Tc-99m-sestamibi and 2' -deoxy-2' -[F-18] fluoro-D-glucose uptake. AT(1) receptor stimulation did not exert any profound effects in quiescent SK-UT-1 cells. The effects induced by Ang II acting on AT(2) receptors were time-dependent. A short, 3 - 6 h lasting stimulation promotes differentiation, i.e increases in the mRNA- and protein levels of SM22 alpha and calponin, whereas a sustained stimulation for 48 h activates the intrinsic apoptotic pathway, as evidenced by reduced cell numbers, down-regulation of the anti-apoptotic Bcl-2 protein and increased levels of the Bax protein and cleaved caspase-3. The effects were reversed by the PPAR gamma antagonist, GW 9662, clearly implying a PPAR gamma-dependent mechanism. Our results also demonstrate a co-localisation of the AT(2)-receptor interacting protein, ATIP1, and the PPAR gamma in nuclei of SK-UT-1 cells and an accumulation of ATIP1 in the nuclear fraction in response to AT(2) receptor stimulation. The regulation of the differentiation and apoptosis via the AT(2) receptor favours an important functional role of this receptor in quiescent, slow-cycling SK-UT-1 cells and provides the rationale for the use of AT(1) receptor antagonists for the treatment of human leiomyosarcomas.