Secreted Cyclophilin A, a Peptidylprolyl cis-trans Isomerase, Mediates Matrix Assembly of Hensin, a Protein Implicated in Epithelial Differentiation

Hu Peng,Soundarapandian Vijayakumar,Cordelia Schiene-Fischer,Hui Li,Jeffrey M Purkerson,Miroslav Malesevic,Jürgen Liebscher,Qais Al-Awqati,George J Schwartz

doi:10.1074/jbc.m808964200

Abstract

Hensin is a rabbit ortholog of DMBT1, a multifunctional, multidomain protein implicated in the regulation of epithelial differentiation, innate immunity, and tumorigenesis. Hensin in the extracellular matrix (ECM) induced morphological changes characteristic of terminal differentiation in a clonal cell line (clone C) of rabbit kidney intercalated cells. Although hensin is secreted in monomeric and various oligomeric forms, only the polymerized ECM form is able to induce these phenotypic changes. Here we report that hensin secretion and matrix assembly were inhibited by the peptidylprolyl cis-trans isomerase (PPIase) inhibitors cyclosporin A (CsA) and a derivative of cyclosporin A with modifications in the d-Ser side chain (Cs9) but not by the calcineurin pathway inhibitor FK506. PPIase inhibition led to failure of hensin polymerization in the medium and ECM, plus the loss of apical cytoskeleton, apical microvilli, and the columnar epithelial shape of clone C cells. Cyclophilin A was produced and secreted into the media to a much greater extent than cyclophilins B and C. Our results also identified the direct CsA-sensitive interaction of cyclophilin A with hensin, suggesting that cyclophilin A is the PPIase that mediates the polymerization and matrix assembly of hensin. These results are significant because this is the first time a direct role of peptidylprolyl cis-trans isomerase activity has been implicated in the process of epithelial differentiation.

Highlights

In this study, using cell lines derived from rabbit kidney collecting ducts, we show that cyclosporin A (CsA) regulates the extracellular matrix assembly of hensin and the differentiation features of kidney intercalated cells by inhibiting peptidylprolyl cis-trans isomerase (PPIase) activity
Inhibitors of PPIase Activity Prevent the Development of Differentiated Phenotype in Clone C Cells—We showed previously that an immortalized clonal cell line derived from ␤-intercalated cells of the rabbit kidney exhibits distinctly different phenotypes when seeded at subconfluent density and superconfluent density [7]
Our results have demonstrated that a specific PPIase inhibitor, Cs9, and CsA inhibited the development of a fully differentiated phenotype of clonal cell line (clone C) cells by inhibiting the polymerization of hensin in the extracellular matrix (ECM)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Stock cultures of clone C of ␤-intercalated cells established from rabbit kidney cortex were maintained at 32 °C [13, 14]. Conditioned Media Preparation and Density Gradient Analysis—Clone C cells were seeded at high density and cultured in the presence or absence of 10 ␮M CsA, 20 ␮M Cs9, or 500 nM FK506 for 2 days at 40 °C and washed with phosphate-buffered saline and cultured in serum-free culture medium for another 6 h. Conditioned medium from both the apical and basal compartments of the Transwell௡ filters from each sample was collected after 6 h and concentrated with Centricon௡ centrifugal filter units (Millipore, Billerica, MA), and protein concentration was determined using a BCA assay (Pierce). The Kruskal-Wallis test was used to compare multiple groups

RESULTS

High density

Cell treatment n Height p value

High Molecular Mass Fractions of

DISCUSSION