Epithelium; Neuropeptides; Ophthalmology; Trabecular Meshwork; Chloride Channels
Major research projects being conducted in my laboratory:
Project # 1 The ciliary epithelium as a novel endocrine secretory epithelium:
The secretion of aqueous humor and the regulation of intraocular pressure are fundamental physiologic processes for the integrity and normal function of the mammalian eye. The ciliary epithelium, a bilayer of secretory neuroepithelial cells, is the site of synthesis of aqueous humor (inflow system), and it regulates its secretion by the coordinated action of a complex transport system that involves ion channels and active transporters. The aqueous humor fluid leaves the eye through the trabecular meshwork and the Schlemm channel (outflow system). A balance between the rates of secretion and drainage of the aqueous humor determines the level of intraocular pressure.
In Primary Open Angle Glaucoma, the most prevalent form of glaucoma, an obstruction of the outflow system leads to an abnormal increase in eye pressure, and with time, to blindness. The most effective way to lower intraocular pressure is by targeting the inflow system with drugs that reduce the rate of secretion of aqueous humor. Thus, an understanding of the biology of the ciliary epithelium, at the cellular and molecular levels, is essential in our knowledge of how intraocular pressure is regulated in normal and disease processes.
Specialized Terms: Chloride Channels of the ClC Family; Construction of Subtractive Libraries; Gene Expression and Regulation of Neuropeptides and Hormones; Gene Expression and Regulation of TIGR; Regulatory Peptides With Hypertensive And/or Hypotensive Act; Steroid Metabolism and Regulation of Gene Expression of 17bH
Extensive Research Description
The ocular ciliary epithelium as a novel endocrine secretory epithelium: The secretion of aqueous humor and the regulation of intraocular pressure (IOP) are physiologically and pathophysiologically important processes for the integrity and normal function of the mammalian eye. Aqueous humor provides nutrients and oxygen to the avascular anterior segment of he eye, and sustains inflation of the globe, ensuring normal visual function. The ciliary epithelium, a bilayer of secretory neuroepithelial cells, is the site of synthesis of aqueous humor (inflow system), and regulates its secretion by the coordinated action of a complex transport system involving ion channels and active ion secretion, from the stromal to the aqueous-humor surface, followed by osmotic water movement. The aqueous humor fluid leaves the eye through the trabecular meshwork and the Schlemm channel (outflow system). A balance between the rates of secretion and drainage of the aqueous humor determines the level of IOP. Clarifying the mechanisms and regulation of aqueous humor formation can lead to novel strategies for lowering IOP, the only intervention known to retard the onset and progression of blindness in glaucoma. In glaucoma, elevated IOP is one of the strongest known risk factors. IOP elevation in human glaucoma results from an obstruction of the outflow system leading to an abnormal increase in eye pressure, death of retinal ganglion cells and loss of vision. The most effective way to lower IOP is by targeting the inflow system with drugs that reduce the rate of secretion of aqueous humor. Thus, an understanding of the biology of the ciliary epithelium, at the cellular and molecular levels, is essential in our knowledge of how IOP is regulated in normal conditions and in glaucoma. Our laboratory has identified a large number of genes relevant to the cell and molecular functions of the human ciliary epithelium. The information of over 300 independent subtracted ESTs (we called CBS clones for Ciliary Body Subtracted) is now deposited and registered in the National Eye Institute Bank (NEIBank), a repository database of the genes expressed in distinct tissues of the human eye on the project website http://neibank.nei.nih.gov). We have proposed that the neuroendocrine peptides and hormones processed by the ciliary epithelium, serve to establish cross-talk communication, according to classical endocrine, paracrine and autocrine modes of action, with target tissues including the vascular endothelium, the ciliary muscle and the cells in the outflow pathway, to regulate IOP.
Current work in our laboratory focuses on the hypotensive function of natriuretic peptides in the eye. We found that natriuretic peptides acting on natriuretic peptide receptors type B in the nonpigmented ciliary epithelium, blocked the activity of the Na+/H+-exchanger, a cellular sensor of cell volume and intracellular pH. The predicted net result of the Na+/H+-exchanger inhibition is the reduction of aqueous humor secretion and IOP. This interpretation is consistent with two sets of independent studies indicating that: 1) inhibitors of the Na+/H+-exchanger; and 2) natriuretic peptides, elicit ocular hypotensive effect by lowering IOP in experimental animals. We do not preclude that this mechanism might be similar in the outflow system where NP receptors are also present (Figure?.) These studies open a great opportunity to consider inhibitors of the Na+/H+-exchanger in the inflow and outflow systems as a key gene target to lower IOP in glaucoma.
Additional studies are under way to elucidate the intracellular signaling pathway of the natriuretic peptide receptors and other neuropeptide receptors including somatostatin receptors that lead to the attenuation of the Na+/H+-exchanger. Glaucoma genes: structure-function relationship Seven chromosomal loci (GLC1A-GLC1G) have so far been implicated in Primary Open Angle Glaucoma, of which three genes, Myocilin (MYOC) on GLC1A (1q23), Optineurin (OPTN) on GLC1E (10qp14) and WDR36 on GLC1G (5q22.1) have been identified.
At present our work focus on myocilin a 55-57kDa glycoprotein of unknown function, originally identified in cultured trabecular meshwork cells upon induction with glucocorticoids. MYOC was independently cloned from the human ciliary body and the retina. MYOC is mutated in certain forms of glaucoma, including Juvenile open angle glaucoma and Primary-open angle glaucoma.
The gene consists of three exons. Exon I, encodes the amino-terminal region of myocilin and contains a cleavable signal peptide at position 32, and a leucine-zipper like domain with leucine and arginine repeats along in an alpha-helix conformation. Exon II, encodes the central region of the protein a linker between the N- and C-terminal regions. Finally, exon III is homologous to olfactomedin, an extracellular matrix protein rich in the olfactory neuroepithelium. The leucine-zipper-like domain of myocilin shares low homology (20-28%) with the ezrin/radixin/moesin (ERM) family of proteins, contractile proteins (smooth muscle myosin), and transcription factors lacking the basic DNA binding region. The ERM proteins crosslink actin filaments with plasma membrane. So far, the majority of the mutations identified in MYOC, in glaucoma patients, have been found along the olfactomedin-like domain (Figure ). Recent studies, in collaboration with Dr. Julio Escribano (Universidad de Castilla-La Mancha, Albacete, Spain), have revealed that myocilin undergoes an intracellular endoproteolytic cleavage between amino acids Arg226 and Ile227 . This processing predicts the production of two fragments which are co-secreted with the nonprocessed myocilin protein. We proposed that inhibition of the normal processing by myocilin when is mutated may explain in part the pathogenesis of glaucoma.
Our work on MYOC focuses on the structural motifs present in myocilin, and on their involvement on multiple molecular interactions with other proteins. Furthermore, we are interested in determining how specific mutations in myocilin leading to glaucoma can affect the interaction with other interacting proteins. Recent studies have shown that myocilin interacts in vitro with extracellular matrix (ECM) proteins, including fibronectin and type I collagen. Ongoing work in our laboratory, at Yale, is searching for proteins that interact with myocilin using the GAL-4 based yeast two-hybrid system. We have identified and initially characterized one gene encoding the half-carboxyl terminus of the protein hevin, a secretory glycoprotein and a member of the SPARC/BM-40/ Osteonectin family of extracellular proteins as an interacting protein with myocilin. Interestingly we have found that a specific mutation in myocilin (P370L) inhibits the normal secretion of the half-carboxyl terminus of the protein hevin, suggesting that myocilin establish a regulatory effect on the normal function of other key proteins (Li et al. 2006).
Further studies are underway to determine the exact inhibitory mechanism exerted by distinct myocilin mutations causing glaucoma. These studies open a great opportunity to explore in what extent mutations causing glaucoma alter the normal function of the cell where is expressed. We are interested in finding this in two specific eye-cell types: in the ciliary epithelium, and in the trabecular meshwork. Both cell types are involved in the regulation of IOP. These studies ultimately will lead us to learn the function of myocilin in glaucoma, and therefore to design strategies to revert the abnormal function of the protein when mutated.
1.Molecular interaction between Myocilin and members of the SPARC family of matricellular proteins and the mechanism for mutant myocilin causing glaucoma.
2. Proteomics analysis of the core of myocilin-interacting proteins
- Aroca-Aguilar, JD., Martinez-Redondo, F., Sanchez-Sanchez, F., Coca-Prados, M., Escribano, J. (2009) Functional role of proteolytic processing of recombinant myocilin in self-aggregation. (In press, Invest Ophthalmol Vis Sci.).
- Coca-Prados M. and Escribano J. (2007) New perspectives in aqueous humor secretion and in glaucoma: The ciliary body as a multifunctional neuroendocrine gland. Progress in Retinal and Eye Research 26: 239-262. Review.
- Sánchez-Sánchez, F., Martínez-Redondo, F., Aroca-Aguilar, J.D., Coca-Prados, M., and Escribano, J. (2007) Characterization of the intracellular proteolytic cleavage of myocilin and identification of calpain II as a myocilin processing protease. J. Biol. Chem. 282: 27810-24.
- Li Y., Aroca-Aguilar J.D., Ghosh S., Sanchez-Sanchez F., EscribanoJ., and Coca-Prados M. (2006) Interaction of myocilin with the C-terminal region of hevin. Biochem. Biophys. Res. Commun 339: 797-804.
- Aroca-Aguilar JD, Sanchez-Sanchez, F, Ghosh S, Coca-Prados M and Escribano J (2005) TIGR/MYOC mutations causing glaucoma inhibit the intracellular endoproteolytic cleavage of myocilin between amino acids Arg226 and Ile227. J. Biol. Chem. 280: 21043-21051.
- Fidzinski, P., Salvador-Silva, M., Choritz, L., Geibel, J., and Miguel Coca-Prados, M. (2004) Inhibition of the NHE-1 Na+/H+-exchanger by natriuretic peptides in the ocular nonpigmented ciliary epithelium. Am J Physiol Cell Physiol.287(3):C655-63.