Monday, June 2, 2025
6/2/2025
Role of CLCA1 as a MIF decoy inhibitor in abdominal aortic aneurysms - Macrophage migration inhibitory factor (MIF) is one of the first cytokines reported nearly 60 years ago. Yet, it took 40 years to find out its receptor CD74. CD74 is also called invariant chain that chaperones MHC-II maturation from ER to endosome/lysosome where CD74 gets degraded by cathepsins. CD74 does not contain a signaling component on its short cytoplasmic tail. This nature of CD74 inspired later discoveries that CD74 does not work alone, but forms complexes with CD44 and chemokine receptors CXCR2 and CXCR4. Since we first reported a direct role for MIF in atherosclerosis, and suggested MIF activity in abdominal aortic aneurysms (AAA) at the time of the CD74 report, we have also started searching for novel MIF receptors. Using mouse aortic smooth muscle cells (SMCs) and recombinant MIF as bait, we discovered a 110-kDa MIF-binding protein: chloride channel accessory protein-1 (CLCA-1). We initially thought it might be a new MIF receptor. CLCA1 is a Ca2+-activated chloride channel (CaCC) expressed in airway epithelial cells, where it lowers the energy barriers for ion transportation as a mechanism to enhance mucin expression and respiratory dysfunction. CLCA1 contains three domains: the CLCA-N domain located on the N-terminus with metallo-hydrolase activity that cleaves CLCA1 into two pieces, the 75-kDa N-terminal fragment and 35-kDa C-terminal fragment, the von Willebrand factor type A (vWA) domain within the 75-kDa fragment to engage the channel to increase Ca2+- dependent Cl– current, and the fibronectin type-III (FN-III) domain within the 35-kDa C-terminal fragment with untested function. Although CLCA1 is not a MIF receptor as we wished, we found that the 35-kDa fragment binds to MIF and acts as a decoy inhibitor that blocks MIF-induced macrophage expression of cytokines and chemokines. We did not detect significant correlations between plasma 35-kDa CLCA1 fragments with AAA size or AAA growth rate from an AAA screening trial, but plasma 75-kDa fragment levels correlated strongly with AAA size and growth rate. We revealed deficiency of the 35-kDa fragment in human and mouse AAA lesions. This fragment is expressed in ECs and SMCs from healthy or diseased aortas. In contrast, the 75-kDa fragment is expressed mainly in macrophages. In CaPO4 injury-induced AAA, MIF deficiency (Mif–/–) reduced AAA growth, but CLCA1 deficiency (Clca1–/–) expedited AAA growth. The increased AAA growth in Clca1–/– mice was fully muted in the absence of MIF (Clca1–/–Mif–/–). CLCA1 with mutations at the cleavage site or the protease active site failed to undergo self-cleavage. CLCA1 knock-in at the cleavage site or protease active site increased AAA growth. We hypothesize that, instead of serving as a MIF receptor, CLCA1 uses its C- terminal 35-kDa FN-III domain-containing fragment to act as a MIF decoy inhibitor that binds to MIF, blocks MIF-induced activation of aortic wall inflammatory and vascular cells, and slows AAA growth. We proposed two Aims to examine the decoy inhibitory role of CLCA1 and its 35-kDa FN-III fragment in MIF-induced AAA and to explore CLCA1 fragment-mediated decoy inhibition of MIF activity on macrophages and vascular cells.
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