Abstract 1949 Poster Session II, Sunday, 5/2 (poster 190)

Stress protein induction by sub-lethal cellular insults is associated with protection against subsequent lethal insults. Treatment with the ansamycin antibiotic Herbimycin A induces stress protein expression without causing apparent cellular injury and protects against injury from heat and ATP depletion. The purpose of this study was to determine the pattern of protein chaperone induction by Herbimycin A (H) in renal epithelia and whether the compound induces stress protein expression directly by activation of Heat Shock Transcription Factor (HSF) or through an alternate pathway.

Cells from the porcine kidney proximal tubule line LLC-PK1 were cultured to confluence and treated with H or vehicle (V). Treatment of the cells with H activated HSF to bind the heat shock element (HSE) on gel retardation assay. LLC-PK1 cells were transiently transfected in parallel with a 1440 base pair (bp) fragment of the HSP-72 gene promotor upstream from the transcriptional start site linked to a luciferase reporter gene and with fragments containing sequential deletions from the 5′ end. Herbimycin A increased luciferase activity 7-fold in cells containing the full length construct. The 700 bp fragment (lacking one HSE and several other regulatory elements but retaining one probable HSE) responded to H with a 5-fold increase in luciferase activity. The 220 bp fragment, containing no apparent HSEs, responded to H with a 2.5-fold increase in luciferase activity.

The expression of three chaperones in response to H were assessed after replacement of the compound with normal media. HSP-72 was induced by 2 hours after treatment with H compared with V, increased five-fold by 6 hours and persisted at this level through 24 hours. The cognate HSC-73 was minimally increased by 6 hours after H treatment and doubled by 24 hours. The pattern of HSP-25 induction was similar to HSC-73.

Herbimycin A, then, appears to induce stress proteins in renal epithelia at least in part through activated HSF regulated gene expression, though an alternate pathway may contribute. Furthermore, H causes a differential induction of the chaperones with HSP-72 being rapidly and highly induced, and the constitutively expressed HSC-73 and HSP-25 induced more slowly and with less exuberance. This targetted, pharmacological induction of protein chaperones causing differential expression may be useful to evaluate function of specific chaperones and to assist cellular repair from injury.