Expression of alternatively spliced growth factor receptor isoforms in the human trabecular meshwork.
OBJECTIVE
Growth factors act through high-affinity cell surface receptors expressed by target cells and are critical modulators of cell function. Because aqueous humor is known to contain growth factors, these molecules may play a key role in maintaining the normal function of the human trabecular meshwork (HTM). Alternate mRNA splicing is an important mechanism used by cells to generate diverse isoforms of growth factor receptors. Although previous investigators have suggested that HTM cells may express alternative isoforms of several growth factor receptors, there have been no studies to verify these preliminary findings. The objective of this study was to determine whether cultured and ex vivo HTM cells express alternate isoforms of hepatocyte, keratinocyte, and transforming growth factor beta (TGFbeta)-II receptors and to characterize the isoform molecular sequences.


METHODS
To determine whether cells within the HTM express mRNA for alternate isoforms of growth factor receptors, total RNA was isolated from several well-characterized HTM cell lines that were established from donors of various ages and from fresh ex vivo HTM tissues from healthy donors. After cDNA 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 beta receptor II (TGFbetaR-II). Specificity and characterization of the polymerase chain reaction amplification products were determined by nucleic acid 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 meshwork tissues expressed specific mRNA for alternatively spliced isoforms of HGFR, KGFR, and TGFbetaR-II The HGFR alternate isoform contained a 96-bp insert in the C-terminal coding region of the cytoplasmic tyrosine kinase domain. The KGFR alternate isoform is a soluble, truncated form, because it has no transmembrane or cytoplasmic domain as does the normal membrane-associated form. The TGFbetaR-II alternate isoform contained a 75-bp insert in 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 TGFbetaR-II. These alternatively spliced receptor isoforms may be functional within the HTM and may play a critical role in maintaining the normal microenvironment of this important tissue.
Wordinger
R J
RJ
Department of Anatomy and Cell Biology and The North Texas Eye Research Institute, University of North Texas Health Science Center at Fort Worth, 76107-2699, USA.
Clark
A F
AF
Agarwal
R
R
Lambert
W
W
Wilson
S E
SE
D000368:Aged; D000369:Aged, 80 and over; D017398:Alternative Splicing; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D006801:Humans; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics* / Q000378:metabolism; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism*; D051497:Receptor, Fibroblast Growth Factor, Type 2; D000077294:Receptor, Transforming Growth Factor-beta Type II; D017468:Receptors, Fibroblast Growth Factor; D017978:Receptors, Growth Factor / Q000235:genetics* / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D014129:Trabecular Meshwork / Q000378:metabolism*
D017931:DNA Primers; D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; D017978:Receptors, Growth Factor; D018125:Receptors, Transforming Growth Factor beta; D012313:RNA; D019859:Proto-Oncogene Proteins c-met; D051497:Receptor, Fibroblast Growth Factor, Type 2; C065104:keratinocyte growth factor receptor; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II