Signaling Pathways in Airway Mucin Secretion: Roles of Protease-Activated Receptor-2, MARCKS, and Related Chaperone and Cytoskeletal Proteins.

Abstract

<DIV> <P style="TEXT-JUSTIFY: inter-ideograph; MARGIN: 0in 0in 0pt; TEXT-ALIGN: justify"><FONT face="Times New Roman" size=3>LIN, KO-WEI.<B> </B>Signaling Pathways in Airway Mucin Secretion: Roles of Protease-Activated Receptor-2, MARCKS, and Related Chaperone and Cytoskeletal Proteins. (Under the direction of Dr. Kenneth B. Adler.)</FONT> <P style="MARGIN: 0in 0in 0pt"><FONT size=3><FONT face="Times New Roman">&nbsp;</FONT></FONT> <P style="TEXT-JUSTIFY: inter-ideograph; MARGIN: 0in 0in 0pt; TEXT-ALIGN: justify"><FONT size=3><FONT face="Times New Roman"><SPAN>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </SPAN>Hypersecretion of mucin (the major glycoprotein component of mucus) causes airway obstruction and complications in diseases such as asthma, chronic bronchitis, bronchiectasis, and cystic fibrosis. After stimulation by secretagogues (agents which provoke secretion), a series of intracellular signals and protein-protein interactions occur, culminating in exocytosis. Although several secretagogues such as serine proteases and allergens have been identified, receptors for secretagogues have not been completely defined. The general stages of mucin secretion include movement of granules from cytoplasm to cell membrane, docking and fusion of granule membranes to the plasma membrane, and release of the granule contents to the airway lumen</FONT><SPAN style="FONT-FAMILY: Arial">. </SPAN><FONT face="Times New Roman">Myristoylated Alanine-Rich C Kinase substrate (MARCKS) protein, a protein kinase C (PKC) substrate, has been shown to be a key molecule regulating this pathway. However, the precise molecular mechanisms that detail how MARCKS becomes linked to mucin granules and promotes exocytosis are still not clear. This dissertation examines the signaling pathways of mucin secretion from two different views, including signaling receptors and intracellular protein-protein interactions. </FONT></FONT> <P style="TEXT-JUSTIFY: inter-ideograph; MARGIN: 0in 0in 0pt; TEXT-ALIGN: justify"><FONT size=3><FONT face="Times New Roman"><SPAN>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </SPAN>First, studies focusing on the putative role of <SPAN>protease-activated receptor-2 (PAR-2) in mucin secretion are discussed. Data presented in this dissertation demonstrate that PAR-2 receptors are present in primary and immortalized bronchial epithelial cells. A </SPAN>synthetic PAR-2 agonist peptide (SLIGKV-NH<SUB>2</SUB>) elicited a small but statistically significant increase in mucin secretion only at high concentrations. Physiological agonists, trypsin and tryptase, did not stimulate mucin secretion.<B> </B>Knockdown of PAR-2 expression by siRNA blocked the stimulatory effect of the synthetic agonist peptide, but mucin secretion stimulated by human neutrophil elastase was not altered. These findings suggest that since PAR-2 activation only weakly increases mucin secretion by human airway epithelial cells <I>in vitro</I>, PAR-2 probably is not a significant contributor to mucin hypersecretion in inflamed airways. </FONT></FONT> <P style="TEXT-JUSTIFY: inter-ideograph; MARGIN: 0in 0in 0pt; TEXT-ALIGN: justify"><FONT size=3><FONT face="Times New Roman"><SPAN style="COLOR: red"><SPAN>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </SPAN></SPAN><SPAN style="COLOR: black">The second study involved protein-protein interactions among MARCKS, chaperone proteins, and cytoskeletal proteins. Previous findings from this laboratory have indicated that the chaperones heat shock protein 70 (</SPAN><SPAN>Hsp70) and cysteine string protein (CSP) are involved in airway mucin secretion. <SPAN style="COLOR: black">We hypothesized that MARCKS is targeted to mucin granules </SPAN>via interactions with Hsp70 and CSP. Data presented here showed MARCKS, Hsp70, and CSP associate with each other in airway epithelial cells. D</SPAN>irect binding between purified MARCKS and Hsp70 <I>in vitro</I> was demonstrated. Co-transfection of fusion proteins demonstrated that MARCKS binds to Hsp70 directly, and Hsp70 binds to CSP directly, but MARCKS does not bind directly to CSP. Interacting domains of MARCKS with these proteins were also revealed. To investigate interactions of MARCKS and cytoskeletal elements, we looked at the possible involvement of an unconventional myosin isoform, myosin V, which has been implicated in secretory granule transport and exocytosis. Both MARCKS and CSP were shown to interact with myosin V, and these interactions were enhanced in response to stimulation of mucin secretion by the phorbol ester, PMA. These results suggest that MARCKS binding to specific chaperones and to the cytoskeletal protein, myosin V, are involved integrally in the mucin secretion pathway.</FONT></FONT> <P style="MARGIN: 0in 0in 0pt"><FONT size=3><FONT face="Times New Roman">&nbsp;</FONT></FONT></P></DIV>