Extracellular matrix gene alternative splicing by trabecular meshwork cells in response to mechanical stretching.
OBJECTIVE
Elevated intraocular pressure (IOP), sensed as mechanical stretching by trabecular meshwork (TM) cells, triggers extracellular matrix (ECM) remodeling. In addition to changes in gene expression, alternative mRNA splicing may alter ECM protein isoforms. Changes in mRNA expression and alternative splicing of four ECM molecules in response to mechanical stretching of TM cells were investigated.


METHODS
Porcine TM cells were mechanically stretched for 12, 24, or 48 hours. RNA was isolated, and RT-PCR was performed with primers that flanked alternatively spliced domains. PCR products were identified by DNA sequencing. Quantitative RT-PCR (qRT-PCR) was performed with primers positioned within nonspliced and spliced regions of the genes.


RESULTS
Total levels of tenascin C, collagen type XII, and CD44 mRNA were increased, whereas versican mRNA levels were decreased in response to the mechanical stretch. In addition, each of these genes expressed alternate mRNA isoforms. Transcripts containing the fibronectin type III domain D of tenascin C, the long NC3 isoform of collagen type XII, the V1 isoform of versican, and exons v7 and v8 of CD44 all increased in response to mechanical stretching. A novel isoform of collagen type XII was observed that resulted in deletion of two exons, a frameshift, and a premature stop codon. This isoform was expressed only by stretched TM cells.


CONCLUSIONS
These alternative splicing events led to the modulation of potential GAG attachment sites and other ECM-binding motifs. These changes should affect TM cell-ECM and/or protein-protein interactions during the ECM remodeling that occurs coincident with homeostatic restoration of IOP to normal.
Keller
Kate E
KE
Casey Eye Institute, Oregon Health and Science University, Portland, Oregon, USA.
Kelley
Mary J
MJ
Acott
Ted S
TS
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D024082:Collagen Type XII / Q000235:genetics; D005109:Extracellular Matrix / Q000235:genetics*; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D015870:Gene Expression / Q000502:physiology; D018960:Hyaluronan Receptors / Q000235:genetics; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013314:Stress, Mechanical; D013552:Swine; D019063:Tenascin / Q000235:genetics; D014129:Trabecular Meshwork / Q000378:metabolism* / Q000473:pathology; D053675:Versicans / Q000235:genetics
D024082:Collagen Type XII; D016326:Extracellular Matrix Proteins; D018960:Hyaluronan Receptors; D012333:RNA, Messenger; D019063:Tenascin; D053675:Versicans