Characterization and functional activity of thrombin receptors in the human lens.
OBJECTIVE
To investigate the expression of thrombin receptors in the human lens, the activation of downstream signaling pathways, and the ability of thrombin to regulate lens cell growth.


METHODS
Thrombin receptor function in the human lens was determined first by measuring changes in intracellular calcium in response to thrombin and protease-activated receptor-activating peptides (PAR-APs). In the human capsular bag model, cell growth was assessed by phase microscope inspection of the cell coverage of the posterior capsular surface. In the human lens cell line FHL124, it was assessed by [(3)H]thymidine incorporation. Changes in p42/p44 ERK phosphorylation (p-ERK) and protein kinase B (PKB/Akt) phosphorylation (p-Akt) were monitored by Western blot. Reverse transcription-polymerase chain reaction (RT-PCR) applied to isolated lens epithelia and ex vivo capsular bag preparations as well as FHL124 cells determined expression of mRNA for the PARs.


RESULTS
Brief exposures to thrombin (10 nM) and PAR1-AP (10 microM) induced an increase in cytosolic calcium in both anterior and equatorial lens cells, but activating peptides for PAR2, -3, and -4 failed to produce responses. Repeated exposure to thrombin produced a significant increase in cell coverage in the capsular bag model and increased [(3)H]thymidine incorporation into FHL124 cells. In the latter, exposure to thrombin (10 nM) and PAR1-AP (10 microM) induced biphasic increases in the phosphorylation of p42/p44 (p-ERK), with peak responses at 20 minutes and 12 hours. Thrombin also produced a 20-fold increase in p-Akt at 12 hours compared with the control, whereas PAR1-AP (10 microM) induced a much smaller response. PAR1-AP did not induce a significant increase in [(3)H]thymidine incorporation and PAR2-AP, PAR3-AP, and PAR4-AP failed to reproduce any of the thrombin-stimulated effects. mPAR1 and -3 were expressed in native lens cells, and this expression was conserved in ex vivo capsular bag preparations as well as in FHL124 cells.


CONCLUSIONS
This study identifies thrombin receptors coupled to calcium, ERK, and Akt signaling that modulate growth in native lens tissue and cultured cells, and it appears that the PAR1 subtype is mainly responsible. PAR3 mRNA was also detected, but the receptor itself, if present, was not coupled to the above signaling elements.
James
Colin
C
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Collison
David J
DJ
Duncan
George
G
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation; D004847:Epithelial Cells / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism*; D008875:Middle Aged; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism; D018179:Receptors, Thrombin / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D013917:Thrombin / Q000494:pharmacology
D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; C105295:protease-activated receptor 3; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D013917:Thrombin; D002118:Calcium