Keratinocyte Secretion of Cyclophilin B via the Constitutive Pathway Is Regulated through Its Cyclosporin-Binding Site

Abstract

Cyclophilin B (CypB) is an endoplasmic reticulum (ER)-resident member of the cyclophilin family of proteins that bind cyclosporin A (CsA). We report that as in other cell types, CypB trafficked from the ER and was secreted by keratinocytes into the media in response to CsA. Concentrations as low as 1 p of CsA induced secretion of CypB. Using brefeldin A, we showed that CypB is secreted from keratinocytes via the constitutive secretory pathway. We defined that substitution of tryptophan residue 128 in the CsA-binding site of CypB with alanine resulted in dissociation of CypBW128A-green fluorescent protein (GFP) from the ER. Photobleaching studies revealed a significant reduction in the diffusible mobility of CypBW128A-GFP compared with CypBWT-GFP, consistent with redistribution of CypBW128A-GFP into secretory vesicles disconnected from the ER/Golgi network. Furthermore, CsA significantly decreased the mobility of CypBWT-GFP but not CypBW128A-GFP. These studies demonstrate that therapeutically relevant concentrations of CsA regulate secretion of CypB by keratinocytes, and that a key residue within the CsA-binding site of CypB controls retention of CypB within the ER and regulates entry into the secretory pathway. As keratinocytes express CypB receptors (CD147) and CypB exhibits chemotactic properties, these data have implications for the therapeutic effects of CsA in inflammatory skin disease.

Depletion of Cyclophilins B and C Leads to Dysregulation of Endoplasmic Reticulum Redox Homeostasis

“…Neither CypB nor CypC contain a "KDEL-like" ER localization signal and previous reports have variously attributed a localization signal on CypB to its C-terminal 10 residues (45) or to a Trp residue within the CsA binding site (46). Consistent with the latter finding, it has been reported that CsA treatment induces CypB secretion from the ER to the media in HeLa and BHK cells (27).…”

“…Neither protein possesses a canonical ER localization sequence, but CypB retention in the ER has been suggested to be conferred by a novel C-terminal motif (VEKP-FAIAKE) (45). Alternatively, because CsA treatment induces CypB (27) and, as shown here, CypC secretion, it has been suggested that ER localization of CypB is regulated through its CsA binding site (46). However, mutations within either region did not substantially alter its ER distribution.…”

Secreted Trypanosome Cyclophilin Inactivates Lytic Insect Defense Peptides and Induces Parasite Calcineurin Activation and Infectivity

“…The mechanism of CypA secretion is largely unknown, although it may involve vesicular transport (40). The related cyclophilin B can also be secreted via targeting through the endoplasmic reticulum with an N-terminal signal sequence (41,42). It is unknown if Cyp19 is destined for secretion using an N-terminal signal sequence, although it possesses a 10-residue N-terminal region not found in human CypA (Fig.…”

Cyclophilin B Attenuates the Expression of TNF-α in Lipopolysaccharide-Stimulated Macrophages through the Induction of B Cell Lymphoma-3

“…Although cytosolic, CyPA can be secreted by macrophages and vascular smooth muscle cells in response to LPS and oxidative stress (14,15). Unlike CyPA, CyPB is constitutively secreted by tissueresident cells (e.g., fibroblasts, chondrocytes, and keratinocytes) and accumulates within the extracellular matrix (16)(17)(18). Importantly, it is released intact by heparanase and matrix metalloproteinases (MMPs), suggesting that it can participate in tissue inflammation.…”