Rickettsia rickettsii infection protects human microvascular endothelial cells against staurosporine-induced apoptosis by a cIAP(2)-independent mechanism.
BACKGROUND
Manipulation of host cell death is an important determinant of the outcome of an infection. Here, we investigate whether Rickettsia rickettsii-infected host endothelial cells resist the effects of staurosporine, a potent inducer of apoptosis, and we explore the mechanisms underlying the anti-apoptotic effect of infection.


METHODS
Human microvascular endothelial cells infected with R. rickettsii for 24 or 48 h were challenged with staurosporine. The extent of apoptosis was evaluated with flow cytometry. mRNA and protein expression levels were determined by use of microarray or polymerase chain reaction and immunoblotting, respectively.


RESULTS
Staurosporine-induced apoptosis in endothelial cells infected for 24 and 48 h was significantly reduced, compared with simultaneously treated uninfected cells. A microarray of human genes involved in apoptosis and polymerase chain reaction analyses revealed increased steady-state mRNA expression of cIAP2 (a member of the inhibitor-of-apoptosis family of proteins) at 24 h after infection. The levels of cIAP2 protein (+/-SD) in infected cells were 3.5 +/- 1.7 -fold and 2.3 +/- 1.2 -fold higher than that in uninfected control cells at 24 and 48 h after infection. Nucleofection of human-specific cIAP2-targeted siRNA resulted in inhibition of protein expression by > or = 50% but had no effect on infection-induced protection against apoptosis.


CONCLUSIONS
R. rickettsii-induced expression of cIAP2 in host endothelial cells is likely not a major contributor to protection against staurosporine-induced cell death.
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Jeremy R
JR
Bechelli
Elena
E
Rydkina
Punsiri M
PM
Colonne
Sanjeev K
SK
Sahni
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; D016923:Cell Death / Q000187:drug effects; D002522:Chlorocebus aethiops; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000172:deficiency / Q000235:genetics*; D008833:Microcirculation / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D012284:Rickettsia rickettsii / Q000187:drug effects; D012373:Rocky Mountain Spotted Fever / Q000188:drug therapy / Q000503:physiopathology*; D019311:Staurosporine / Q000494:pharmacology*; D044767:Ubiquitin-Protein Ligases; D014709:Vero Cells
D004791:Enzyme Inhibitors; D051034:Inhibitor of Apoptosis Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C494832:BIRC3 protein, human; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; D044767:Ubiquitin-Protein Ligases; D019311:Staurosporine