Novel splice variants of human tenascin-C mRNA identified in normal and bullous keratopathy corneas.
OBJECTIVE
Pseudophakic/aphakic bullous keratopathy (PBK/ABK) human corneas accumulate an extracellular matrix glycoprotein tenascin-C (TN-C), an important modulator of cell adhesion and migration. Here, the purpose was to identify specific TN-C mRNA splice variants in normal and PBK/ABK human corneas.


METHODS
Conventional and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) with primers to alternatively spliced (insertional) and constitutive fibronectin type II-like repeats of TN-C was used. Splice variants were identified by cloning and sequencing of RT-PCR products or by Southern blot analysis.


RESULTS
The majority of corneal TN-C mRNA species corresponded to relatively small forms of the protein. Four previously unidentified TN-C mRNA splice variants were found in normal and PBK/ABK corneas that contained insertional repeats A1+A2+B+D, A1+A2+D, A1+B+D, or A1+D. Variants with insertional repeats A1+A2 or A1, previously described in mouse and rat, were also identified in human corneas. Semiquantitative RT-PCR showed that novel TN-C mRNA variants were dramatically elevated in PBK/ABK compared to normal corneas.


CONCLUSIONS
TN-C protein was found in PBK/ABK but not in normal corneas; however, both normal and diseased corneas contained mRNA for 15 different TN-C isoforms. PBK/ABK corneas had elevated levels of six relatively small TN-C mRNA variants including five novel ones. These specific isoforms may adversely affect adhesion and migration of corneal cells thus contributing to the exacerbation of PBK/ABK.
Saghizadeh
M
M
Ophthalmology Research Laboratories, Burns and Allen Research Institute, Cedars-Sinai Medical Center, UCLA Medical School Affiliate, Los Angeles, California 90048, USA.
Khin
H L
HL
Bourdon
M A
MA
Kenney
M C
MC
Ljubimov
A V
AV
D017398:Alternative Splicing / Q000235:genetics*; D015139:Blotting, Southern; D003315:Cornea / Q000737:chemistry*; D003316:Corneal Diseases / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D019063:Tenascin / Q000235:genetics*; D014158:Transcription, Genetic
D017931:DNA Primers; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D019063:Tenascin