"Novel Signaling Pathways Regulated by Prostanoid Receptors in Human Ciliary Smooth Muscle Cells¡¨

ABSTRACT
Glaucoma is a disease in which the death of retinal ganglion cells causes a loss of peripheral vision and, in many cases, total blindness. A major treatment option for glaucoma patients is the use of synthetic analogs of the prostaglandins, which are endogenous local signaling molecules derived from arachidonic acid by the action of the cyclooxygenases. These drugs are effective in the treatment of glaucoma because they reduce intraocular fluid pressure by promoting the outflow of aqueous humor through the ciliary muscle. Animal studies show that these drugs act on E and F prostanoid receptors (EP and FP, respectively) in ciliary muscle and can increase aqueous outflow by triggering tissue remodeling processes that expand intercellular space. To investigate the signaling mechanisms that could potentially couple prostanoid receptor activation to tissue remodeling, we have established primary cultures of human ciliary smooth muscle (HCSM) cells from fetal eye tissue explants. The cells that grew out of these explants were characterized by positive immunoreactivity for alpha-smooth muscle-actin, a specific marker for smooth muscle tissue. These cells were shown by RT-PCR analyses to express mRNAs encoding the EP2 and FP receptors. In additional ongoing studies, we are using selective agonists and signaling inhibitors to examine the second messenger activation, signal transduction, and regulated gene expression that occurs in prostaglandin-stimulated HCSM cultures. To date, we have determined that stimulation of the cells with prostaglandin E2 (PGE2), the endogenous ligand for the EP receptors, elicits a dose-dependent accumulation of cyclic AMP, the major second messenger associated with EP2 receptor activation. Likewise, we have observed the activation of the second messenger inositol trisphosphate (IP3) in HCSM cells stimulated with prostaglandin F2£\ (PGF2£\), which binds the FP receptor. We have also observed that stimulation of HCSM cells with PGF2£\ up regulates the protein levels of hypoxia-inducible factor-1£\ (HIF-1£\) and early growth response factor-1 (EGR-1), which are transcription factors that mediate physiological responses to oxygen deprivation and tissue wounding, respectively. Additionally, PGF2£\ stimulation of HCSM cells induces reporter gene activity of plasmids containing either the HIF response element (HRE) or the antioxidant response element (ARE). These signaling effectors, particularly HIF-1£\/HRE and EGR-1, are known to regulate tissue remodeling processes in other physiological settings and are potentially involved in the expansion of intercellular space observed in the ciliary muscle. The regulation of ARE promoter activity in HCSM cells indicates the possibility of additional therapeutically relevant signaling and gene expression mechanisms activated by prostanoid stimulation. Taken together, these findings indicate that cultured HCSM cells express functional prostanoid receptors that are coupled to novel signaling pathways that may underlie the therapeutic effect of antiglaucoma drugs.