We previously found that laminin-2 (LN-2) is involved in mouse bronchial myogenesis. Our ongoing studies indicate that the synthesis of LN-2 is mechano-sensitive. In recent studies we identified a new set of stretch- referred to as Tension Induced/Inhibited Proteins (TIPs). One of them, TIP-1, is induced by stretch and promotes smooth muscle (SM) myogenesis. Our preliminary data indicate that TIP-1 expression in lung embryonic mesenchymal cells is induced by LN-2. Preliminary data also suggest that TIP-1 upregulates the level of the GTPase Cdc42 and that Cdc42 stimulates SM myogenesis. Based on this we hypothesize that LN-2 regulates myogenesis by a signaling pathway that involves TIP-1, depends on mechanical stimuli and engages Cdc42 as a downstream effector. To test this hypothesis we propose three aims: Aim #1 - to determine the role of Cdc42 in lung myogenesis. Aim #2 - To determine whether LN-2 mediated bronchial myogenesis involves TIP-1. This aim will have two subaims: A. Effect of LN-2 on TIP-1 expression. B. Effect of blocking TIP-1 on LN-2-mediated SM myogenesis. Aim #3 - to determine whether Cdc42 acts downstream of TIP-1 in LN-2/TIP-1 stimulation of bronchial myogenesis. This aim will have two subaims: A. Effect of LN-2 and TIP-1 on Cdc42 expression. B. Effect of blocking Cdc42 on LN-2/TIP-1-mediated myogenesis. To address these aims we will use cell and organ cultures plus functional probes to stimulate or inhibit Cdc42, LN-2 and TIP-1 level/activity. Among the probes we will use purified LN-2, LN-2, Cdc42 and TIP-1 expression constructs and LN-2, Cdc42 and TIP-1 RNA interference constructs. Occurrence of myogenesis will be determined by a series of SM markers and by visualization of the entire bronchial tree in whole lung explants after immunostaining for SM a-actin. The proposed studies will unravel a previously unexplored molecular mechanism whereby tension-induced LN-2 and TIP-1 contribute to the development of lung bronchial muscle. We anticipate, therefore that this project will advance our understanding of lung development as well as diseases caused by, or resulting from abnormal SM quantity, distribution and function.

R37HL048730

1992-09-01

2013-07-31