β1 and β3 integrins use two distinct pathways to regulate CLAN formation

October 1, 2007

Take-home message: New discoveries about the signaling pathways regulating cross-linked actin network (CLAN) formation could in the future lead to new therapeutic approaches for steroid-induced glaucoma. CLANs are found in glaucomatous trabecular meshwork cells as well as steroid-treated cell and eye organ cultures, thus the discovery of two distinct signaling pathways is a preliminary step toward targeting of specific proteins associated with steroid-inducedglaucoma.

Keypoints:

Madison, WI-Researchers have identified two distinct signaling pathways that regulate formation of cross-linked actin networks (CLANs), a discovery that may help identify potential new targets for therapies to treat steroid-induced glaucoma, said Mark S. Filla, PhD, a researcher in the pathology and laboratory medicine department, University of Wisconsin, Madison.

CLANs are found in glaucomatous trabecular meshwork cells as well as steroid-treated cell and eye organ cultures and may decrease aqueous outflow and increase IOP. It has been suggested that one way in which CLANs impede outflow is by perturbing the normal contraction and relaxation of the trabecular meshwork, which in turn affects the ability of the cells to regulate outflow, Dr. Filla said. The study of CLAN formation, he added, is an extension of the theory of cytoskeletal involvement in the regulation of outflow, which shows that signaling from the extracellular matrix regulates the structure of the trabecular meshwork.

Although CLAN structures have not been directly correlated with causing steroid-induced glaucoma, there seems to be some sort of association, either causal or an indirect effect of the disease itself, Dr. Filla said. But if CLANs play a direct role in development of the disease, it might one day be possible to develop therapies targeting the proteins found to be involved in the signaling pathways that regulate CLAN formation.

He and colleagues found that CLAN formation is regulated by integrins and involves cooperative signaling between the β1 and β3 integrins. When signaling is activated for both of these integrins, CLAN formation increases significantly.

The researchers have been attempting to determine which signaling transduction molecules are important in regulating the signaling pathway that begins with the integrins and ends with CLANs. They used a series of pharmacologic inhibitors, activating peptides, and antibodies to assess the role of several candidate molecules.

CLAN formation was induced in human trabecular meshwork (HTM) cells by plating cells for 3 hours on fibronectin-coated coverslips in the presence or absence of the β3 integrin-activating antibody AP-5. Constituents of the integrin signaling pathways were determined by plating HTM cells in the absence or presence of the IAP-activating peptide 4NIK (100 and 200 μg/ml), the IAP-blocking antibody B6H12.2 (20 μg/ml), the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (20 μM), the Rac1 inhibitor NSC23766 (20 μM), or the Src inhibitor PPP2 (5 and 20 μM). Cells were fixed and labeled with phalloidin, and the percentage of CLAN-positive cells was determined using fluorescence microscopy.

"We found that two distinct pathways are involved," Dr. Filla said. One begins with β1 integrins and the other with β3 integrins. At some point, the two pathways converge; no data are yet available on where that takes place, although investigators speculate that it occurs downstream of Rac1 and PI3K.

In both pathways, signaling is dependent on Src or an Src-family kinase. PI3K regulates CLAN formation mediated by β1 integrins but not β3 integrins, whereas β3-mediated CLAN formation is Rac1-dependent and involves IAP.

"We've clearly begun to get a handle on just which pathways are involved," Dr. Filla said. "The nice thing about this approach is that not only can you dissect out the pathways, but you can start to identify potential targets that at some point in the future might be used to develop therapies that could be used to treat steroid-induced glaucoma."