Panoply™ Human CALCA Over-expressing Stable Cell Line

The CALCA gene, located on the short arm of human chromosome 11, region 15.2 (11p15.2), exemplifies the complexity and precision of gene expression in eukaryotes. Through tissue-specific RNA alternative splicing, a single transcript can produce two distinct bioactive peptides: calcitonin (CT) and calcitonin gene-related peptide (CGRP). CGRP has shown potential in treating dry eye syndrome (DED), but its mechanism of action remains unclear. Here, researchers found that CGRP expression in the tears of dry eye patients was significantly positively correlated with multiple clinical indicators of dry eye. CGRP expression was also elevated in animal models of dry eye. Exogenous CGRP peptide significantly inhibited corneal tissue cell apoptosis. The CGRP overexpression group also showed reduced levels of corneal tissue cell apoptosis. Reduced corneal tissue apoptosis was associated with the IFN-γ/JAK2/STAT1 pathway. These findings indicate that CGRP (8–37) can simultaneously increase apoptosis, inhibit the expression of specific interferon-stimulated genes (SISGs), and is associated with the activation of the IFN-γ/JAK2/STAT1 pathway.

Here, researchers used a CGRP receptor antagonist (CGRP(8-37)) to intervene in CGRP-overexpressing HCET cell lines (CGRP(8-37) group) to further verify the anti-apoptotic mechanism of CGRP. JC-1 probe detection results showed that the green/red fluorescence intensity of the CGRP(8-37) group was significantly higher than that of the CGRP-OE group (Figure 1A, B). Flow cytometry analysis showed that the proportion of ROS-positive cells in the CGRP(8-37) group was significantly higher than that in the CGRP-OE group (Figure 1C, D). ROS staining microscopy results also showed that the degree of oxidative stress in the CGRP(8-37) group was significantly higher than that in the CGRP-OE group (Figure 1E, F). Annexin V-PE/7-AAD apoptosis detection kit results showed that the proportion of late apoptotic cells in the CGRP(8-37) group was significantly higher than that in the CGRP-OE group (Figure 1G, H). Western blot analysis also confirmed that the expression levels of corneal apoptosis-related proteins cleaved caspase-3 and cleaved PARP1 in the CGRP(8-37) group were significantly higher than those in the CGRP-OE group (Figure 1I and L). Western blot analysis also showed that the expression levels of IFN-γ, p-STAT1, and p-JAK2 in the CGRP(8-37) group were significantly higher than those in the CGRP-OE group (Figure 1J and M). Conversely, Western blot analysis showed that the protein expression levels of c-MAF, IFI44, MX2, RTP4, OAS2, and UBA7 in the CGRP(8-37) group were significantly lower than those in the CGRP-OE group (Figure 1K and N).

Figure 1. CGRP(8-37) activated the IFN-γ/JAK2/STAT1 pathway and increased the level of apoptosis by targeting SISGs. (Hong X, et al., 2025)