Abstract
Purpose: :
MYLK was among the differentially expressed genes in African American ONH astrocytes detected by microarray. In this study, we investigated its expression profile in ONH astrocytes and its physiological role.
Methods: :
ONH astrocytes from 11 Caucasian American normal donor eyes (CA), 12 African American normal donor eyes (AA), 10 Caucasian American donor eyes with glaucoma (CAG) and 6 African American donor eyes with glaucoma (AAG) were used in this study. qRT-PCR measured MYLK mRNA. Western blots detected its different isoforms. PCR detected alternative splicing within the long isoform. Cell migration assay measured migratory properties of the astrocytes. ANOVA was used to test significance.
Results: :
qRT-PCR showed higher MYLK mRNA levels in AA and AAG astrocytes compared to CA and CAG astrocytes (p = 0.0048). Abundant MYLK staining was detected in ONH tissues of AA and AAG. Migration experiments indicate that astrocyte migratory properties correlate with MYLK levels. AA and AAG astrocytes migrated faster than CA and CAG (p = 0.0139). Inhibition of MYLK with ML-7 (10 µM), a specific inhibitor led to slower migration, compared to vehicle (DMSO) treated cells (n = 3, p = 0.03). MYLK has three transcription promoters and multiple alternative splicing sites. Western blots detected three bands at 210, 208 and 130 kDa respectively. MYLK long forms (208-210 kDa) were present in all groups, while MYLK short from (130 kDa) was expressed in AA and CA, with lower levels in CAG, and was not detected in AAG. Alternative splicing occurs in the long MYLK expressed in astrocytes. Using primers for known alternative splicing sites, MYLK isoforms 1 and 2 were detected in all 28 astrocytes cultures from four groups tested, while isoforms 3A and 3B were absent.
Conclusions: :
Our data showed that AA and AAG astrocytes express more MYLK in vitro and in situ. Consistent with the level of MYLK, AA and AAG astrocytes migrate significantly faster. Inhibition of MYLK with ML-7 slowed down astrocytes migration significantly. The MYLK long forms were detected in all four groups, while MYLK short form was absent in AAG and expressed at a lower level in CAG. Overall, our results showed that MYLK exhibited different expression profiles in glaucomatous astrocytes and its kinase activity is necessary for astrocyte migration. Given its location within the known POAG locus GLC1C, our study suggests that MYLK is a potential glaucoma susceptibility gene in AA population.
Keywords: astrocytes: optic nerve head • transcription