Because HCECs show characteristics that distinguish them from RCECs (such as the need for coculture with a feeder layer), we therefore evaluated whether both CI and CIV would exert influences in HCECs similar to those observed in RCECs. To that purpose, HCECs were cultured on CI or CIV to either subconfluence (SC) or complete confluence (C) and then transfected with the α5 promoter bearing constructs −132α5/CAT and −954α5/CAT. As shown in
Figure 4A, CI had little influence, if any, on the activity directed by the α5 promoter contained on the −132α5/CAT plasmid in either subconfluent (CI
SC) or confluent (CI
C) HCECs. Conversely, CIV caused a significant increase in the CAT activity in a cell density–independent manner (2.5- and 2.2-fold increase in subconfluent [CIV
SC] and confluent [CIV
C] HCECs compared with BSA, respectively). When the −132α5/CAT construct was substituted by the −954α5/CAT plasmid, repression of α5 promoter activity was observed using either CI or CIV at any cell density (
Fig. 4B). Quantitative PCR analyses further supported repression of the endogenous
α5 gene transcription by both CI and CIV relative to BSA in HCECs grown to sub- or postconfluence (
Fig. 4C). These CI- and CIV-dependent repressions of endogenous
α5 gene transcription also translated into corresponding reductions in the amount of α5 integrin subunit at the protein level (reduction ranging from 2.5- to 15.5-fold on normalization to actin levels), as revealed by Western blot analyses (
Fig. 4D). The reduced α5 promoter activities observed in HCECs grown on collagens also correlate with reduced DNA-binding interaction of Sp1 (
Fig. 5A, compare lanes 2 and 3) and NFI (
Fig. 5A, compare lanes 6 and 7) by CI and AP-1 by CIV (
Fig. 5A, compare lanes 10 and 12). Interestingly, DNA binding of PAX-6, a transcription factor expressed in the cornea
53 that has been shown to participate to
α5 gene transcription,
54 is also reduced by CIV (
Fig. 5A, compare lanes 14 with 16). However, very little change in the nuclear content of these transcription factors was observed by Western blot, with the exception of a weak decrease in the expression of the AP-1 subunits c-Fos and FosB in cells grown on CI and c-Fos and Fra2 in cells grown on CIV (
Fig. 5B). Therefore, the reduced binding of these TFs to their respective target sites is more likely to be related to changes in their posttranslational status, as previously reported,
49,52 rather than to corresponding changes in their protein concentration. In addition,
α5 gene promoter repression in HCECs (
Fig. 4) was clearly of a lesser amplitude than that observed in RCECs (
Fig. 2), which is also consistent with the less massive alterations in the expression of the transcription factors that drive expression of
α5 gene transcription in HCECs (
Fig. 5) compared with that observed in RCECs (
Fig. 3).