Proliferation of the human urothelium is induced by atypical β₁‐adrenoceptors

Abstract

We wanted to assess whether β-adrenoceptors mediate proliferation in the normal and malignant urothelial cell lines UROtsa and T24, respectively. Urothelial cells were cultured for 24 h in the presence of the β-adrenoceptor agonists isoprenaline (β1/2/3 ), dobutamine (β1 ), salbutamol (β2 ), BRL 37344 (β3 ), CGP 12177 (a partial β-agonist) or β-adrenoceptor antagonists (metoprolol; β1 , propranolol; β1/2 ). Phosphorylation of kinases was screened with a Human Phospho-Kinase Array Kit (R&D systems). Intracellular pathways activated by proliferation of urothelial cells were characterized by incubating cells with the MEK1/2 inhibitor PD 98,059, the p38 kinase inhibitor losmapimod or with the Akt 1/2 kinase inhibitor. Proliferation was assessed with the MTT proliferation assay (ATCC). Western blot and immunocytochemistry were used for detection of the β1 -adrenoceptor. Isoprenaline and dobutamine induced proliferation, while salbutamol and BRL 37344 did not. Dobutamine-induced proliferation was not affected by metoprolol or propranolol but was instead antagonized by CGP 12177 in T24 but not in UROtsa. In response to stimulation with dobutamine, Akt1/2/3 was phosphorylated in UROtsa, while ERK1/2 and p38 were phosphorylated in T24. MEK1/2 inhibition blocked basal and dobutamine-induced proliferation in T24 but only basal proliferation in UROtsa. Losmapimod slightly inhibited basal proliferation in T24 but not dobutamine-induced proliferation. Akt 1/2 inhibitor blocked basal and dobutamine-induced proliferation in UROtsa. Immunocytochemistry and Western blot revealed expression of β1 -adrenoceptors in both urothelial cell lines. The present data show that the urothelium expresses atypical β1-adrenoceptors that activate intracellular kinases inducing urothelial proliferation.

The prostaglandin H2 analog U-46619 improves the differentiation efficiency of human induced pluripotent stem cells into endothelial cells by activating both p38MAPK and ERK1/2 signaling pathways

“…The medium was changed every 2 days, and differentiation efficiency was evaluated on day 11 via fluorescence-activated cell sorting (FACS); cells positive for CD31 expression and for both CD31 and CD144 expression were collected and expanded. For investigations of p38MAPK and ERK1/2 inhibition, the inhibitors (10 μM Losma, an inhibitor of p38MAPK [ 21 ], and/or 5 μM SCH , an inhibitor of ERK1/2 [ 22 ]) were added to the differentiation medium 30 min before CHIR, VEGF/TGFβ1/EPO, or U46619 treatment was initiated.…”

“…Here, we investigated the pathways involved in hiPSC-EC differentiation to determine whether our protocol could be made even more efficiently by targeting the p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling pathways, which have been shown to contribute independently to the EC differentiation of pluripotent stem cells [ 19 , 20 ]. Thus, we investigated the temporal dependence of our hiPSC-EC differentiation protocol on these signaling pathways by treating the cells with selective inhibitors of p38MAPK (losmapimod [Losma]) [ 21 ] or ERK1/2 (SCH772984 [ SCH ]) [ 22 ] during differentiation stages. We found that our enhanced protocol can not only be used to generate ECs from the non-disease hiPSCs, but also cells of patients whose disease or disease symptoms have been linked to endothelial dysfunction, such as type 2 diabetes and atherosclerosis in patients with Hutchinson-Gilford progeria syndrome [ 23 – 25 ], which have not been achieved with high differentiation efficiency.…”

Autonomic Receptor‐mediated Regulation of Production and Release of Nitric Oxide in Normal and Malignant Human Urothelial Cells

“…Once the cells reached a confluency of 70%, they were treated with either phosphate‐buffered saline (PBS; serving as control), the beta‐adrenoceptor agonist dobutamine (1 × 10 −4 M; Tocris Bioscience, Bristol, UK), the alpha‐adrenoceptor agonist phenylephrine (5 × 10 −4 M; Sigma‐Aldrich), the non‐selective muscarinic agonist methacholine (1 × 10 −4 M; Sigma‐Aldrich), the P2 purinoceptor agonist adenosine 5′‐triphosphate (ATP; 5 × 10 −5 M; Sigma‐Aldrich), the P1 purinoceptor agonist adenosine (5 × 10 −5 M; Sigma‐Aldrich), the non‐selective muscarinic antagonist atropine (1 × 10 −4 M; Sigma‐Aldrich), the alpha‐adrenoceptor antagonist phentolamine (5 × 10 −5 M; Sigma‐Aldrich), the β 1/2 ‐selective adrenoceptor antagonist propranolol (1 × 10 −5 M; Sigma‐Aldrich) or the β 3 ‐selective adrenoceptor antagonist L‐748,337 (1 × 10 −5 M; Tocris Bioscience). The chosen drug concentrations were based on pilot studies and previous viability data from MTT cell proliferation assays . After 24 hr, the cells were treated with 1,2‐diaminoanthraquinone (DAQ; 1.5 μg/mL) for 30 min.…”

Biofabrication of a Tubular Model of Human Urothelial Mucosa Using Human Wharton Jelly Mesenchymal Stromal Cells

“…Although some of these models showed good preclinical and clinical results, the ideal substitute of the human UM has not been described to date. One of the main challenges in tissue engineering is finding an appropriate cell source able to provide large cell quantities [ 7 ], since many cell types, especially human epithelial cells, are difficult to culture and show very low proliferation rates [ 14 , 15 , 16 ]. One of the possible alternative cell sources for the generation of human cell cultures is the umbilical cord Wharton’s jelly [ 17 ].…”