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

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 pM 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 CypB(W128A)-green fluorescent protein (GFP) from the ER. Photobleaching studies revealed a significant reduction in the diffusible mobility of CypB(W128A)-GFP compared with CypB(WT)-GFP, consistent with redistribution of CypB(W128A)-GFP into secretory vesicles disconnected from the ER/Golgi network. Furthermore, CsA significantly decreased the mobility of CypB(WT)-GFP but not CypB(W128A)-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.