Activating transcription factor 3 (ATF3) responds to diverse cellular stresses and can be activated by several important signal transduction pathways.
74,75 It is well established that following stress, downregulation of mammalian target of rapamycin (mTOR) initiates autophagy.
76,77 Recently, Sirolimus, an mTOR inhibitor, was demonstrated to decrease ATF3 expression in 3D organotypic skin culture models.
78 Based on this finding, the prediction is that compromised autophagy will result in an increase in ATF3 expression. Indeed,
Atf3 was one of the highest upregulated genes in the beclin 1
+/− scRNA-seq data (
Fig. 6F,
Supplementary Table S3). ATF3 expression in wild-type mice was most prominent in the nuclei of limbal epithelial basal cells (
Fig. 9A). Such expression was markedly enhanced in the limbal epithelial basal cells from the beclin 1
+/− mice (
Fig. 9A), with a significant increase in ATF3+ cell number in limbal epithelia of beclin 1
+/− mice (
Fig. 9B), and is consistent with the scRNA-seq data. Although ATF3 antibodies tested under different fixation conditions failed give a consistent nuclear staining pattern with human cornea sections, immunoblots from lysates of human limbal and corneal epithelial cells revealed strong expression of ATF3 in the limbal epithelial cells (
Fig. 9C). Moreover, real-time quantitative PCR of the total RNA isolated from the cornea showed increased mRNA transcript level for ATF3 in beclin 1
+/− mice compared with the wild-type littermate controls (
Fig. 9D). In a mouse model of heart failure, ATF3 reduced autophagy via suppression of the beclin-1–dependent pathway.
79 This negative regulation of autophagy via ATF3 is in line with the present findings of an increase in ATF3 in the limbal epithelium of mice with compromised autophagy (
Fig. 9A). In addition to stress response, ATF3 has been demonstrated to both positively and negatively affect proliferation in a variety of oncogenic settings.
80–83 We have shown that autophagy plays a positive role in corneal epithelial proliferation,
26 and thus wanted to examine how modulation of ATF3 affects proliferation in these cells. Using a genetic approach, we treated hTCEpi cells with siATF3 and noted a significant increase in cell growth compared with the control siRNA (
Fig. 9E). This suggests that in the context of normal, resting corneal epithelial cells, ATF3 dampens proliferation and may, in part, contribute to the maintenance of quiescence in the stem cell–enriched limbal basal cells. Furthermore, the marked increase in ATF3 expression in limbal epithelium of beclin1
+/− mice may contribute to the delayed corneal epithelial wound-healing response observed in these autophagy-compromised mice.
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