Differential inhibition of HSP72 and HSP25 produces profound impairment of cellular integrity.
To test a putative cause and effect relationship between heat-shock protein (HSP) expression and response to renal cell injury, HSP72 and HSP25 were differentially inhibited in LLC-PK1 cells by means of transcription factor decoy and short interference RNA (siRNA). Cellular injury was assessed by solubilization of NaK ATPase (S-NaK). An exonuclease-resistant, ethylene glycol-bridged, circular oligonucleotide decoy for heat-shock transcription factor (HSF)-1, based on the sequence of the porcine heat-shock element, was constructed and validated. It was found that under all experimental conditions, cells had comparable ATP levels; that decoy of unligated or scrambled sequence was ineffective; that HSP72 mRNA and HSP72/HSP25 proteins were significantly reduced in decoy-treated cells; and that the dampened response to HSF activation in decoy-treated, injured cells was accompanied by a substantially amplified loss of cellular integrity (S-NaK was 85% compared with baseline levels). Specific inhibition of HSP72 that used siRNA directed against an inducible porcine HSP72 gene resulted in complete ablation of injury-induced HSP72. Isolated inhibition of HSP72 was also associated with marked NaK ATPase detachment from the cytoskeleton (S-NaK was 135% compared with baseline levels). These studies suggest that an HSF-1 decoy effectively dampens the HSP72/HSP25 response to injury in renal cells; that HSP72 siRNA ablates injury-induced induction of HSP72; and that dampening of the HSP72/HSP25 response and ablation of the HSP72 response are both associated with impaired restitution of cellular polarity.
Riordan
Michael
M
Department of Pediatrics, Yale University, New Haven, CT 06520-8064, USA. mriordan@doctors.org.uk
Garg
Vivek
V
Thulin
Gunilla
G
Kashgarian
Michael
M
Siegel
Norman J
NJ
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D020868:Gene Silencing; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase