Protein Synthesis, Post-Translational Modification, and Degradation
Secreted Cyclophilin A, a Peptidylprolyl cis-trans Isomerase, Mediates Matrix Assembly of Hensin, a Protein Implicated in Epithelial Differentiation*

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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.

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The abbreviations used are: ECM, extracellular matrix; SRCR, scavenger receptor cysteine-rich; SID, SRCR-interspersed domain; ZP, zona pellucida; PPIase, peptidylprolyl cis-trans isomerase; CsA, cyclosporin A; Cyp, cyclophilin; LiE, 6-(3,4-dichlorophenyl)-4-(N,N-dimethylaminoethylthio)-2-phenylpyriidine; Fmoc, N-(9-fluorenyl)methoxycarbonyl; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; RT, reverse transcription; Ni-NTA, nickel-nitrilotriacetic acid; ANOVA, analysis of variance; HD, high density; FKBP, FK506 (tacrolimus)-binding protein.

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G. Fischer and J. Liebscher, unpublished results.

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This work was supported, in whole or in part, by National Institutes of Health Grants DK-20999 (to Q. Al-A.) and DK-50603 (to G. J. S.). This work was also supported by Grant-in-aid 0150138N from the American Heart Association (to G. J. S.) and by Deutsche Forschungsgemeinschaft Grant GK1026 (to C. S.-F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

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Both authors contributed equally to this manuscript.