Expression of alternatively spliced growth factor receptor isoforms in thehuman trabecular meshwork

PURPOSE. Growth factors act through high-affinity cell surface receptors ex pressed by target cells and are critical modulators of cell function. Becau se aqueous humor is known to contain growth factors, these molecules may pl ay a key role in maintaining the normal function of the human trabecular me shwork (HTM). Alternate mRNA splicing is an important mechanism used by cel ls to generate diverse isoforms of growth factor receptors. Although previo us investigators have suggested that HTM cells may express alternative isof orms of several growth factor receptors, there have been no studies to veri fy these preliminary findings. The objective of this study was to determine whether cultured and ex vivo HTM cells express alternate isoforms of hepat ocyte, keratinocyte, and transforming growth factor beta (TGF beta)-II rece ptors and to characterize the isoform molecular sequences. METHODS. TO determine whether cells within the HTM express mRNA for alterna te isoforms of growth factor receptors, total RNA was isolated from several well-characterized HTM cell lines that were established from donors of var ious ages and from fresh ex vivo HTM tissues from healthy donors, After cDN A synthesis, polymerase chain reaction was initiated using specific primers for alternate forms of the following receptors: hepatocyte growth factor ( HGFR), keratinocyte growth factor (KGFR), and transforming growth factor be ta receptor II (TGF beta R-II). Specificity and characterization of the pol y merase chain reaction amplification products were determined by nucleic a cid sequencing. RESULTS. Amplification products of the expected size for the growth factor isoforms were expressed in cell lines and in ex vivo tissues. Nucleic acid sequencing showed that cultured HTM cells and fresh ex vivo trabecular mesh work tissues expressed specific mRNA for alternatively spliced isoforms of HGFR, KGFR, and TGF beta R-II The HGFR alternate isoform contained a 96-bp insert in the C-terminal coding region of the cytoplasmic tyrosine kinase d omain. The KGFR alternate isoform is a soluble, truncated form, because it has no transmembrane or cytoplasmic domain as does the normal membrane-asso ciated form. The TGF beta R-II alternate isoform contained a 75-bp insert i n the N-terminal coding region of the extracellular domain. CONCLUSIONS. In vitro and ex vivo HTM cells express mRNA for alternatively spliced isoforms of HGFR, KGFR, and TGF beta R-II. These alternatively spli ced receptor isoforms may be functional within the HTM and may play a criti cal role in maintaining the normal microenvironment of this important tissu e.