pubmed.ncbi.nlm.nih.gov

Astrocyte apoptosis induced by HIV-1 transactivation of the c-kit protooncogene - PubMed

  • ️Wed Jan 01 1997

Astrocyte apoptosis induced by HIV-1 transactivation of the c-kit protooncogene

J He et al. Proc Natl Acad Sci U S A. 1997.

Abstract

HIV-1 infection of the central nervous system (CNS) frequently causes dementia and other neurological disorders. The mechanisms of CNS injury in HIV-1 infection are poorly understood. Apoptosis of neurons and astrocytes is induced by HIV-1 infection in vitro and in brain tissue from AIDS patients, but the apoptotic stimuli have not been identified. We report herein that HIV-1 infection of primary brain cultures induces the receptor tyrosine kinase protooncogene c-kit and that high levels of c-Kit expression are associated with astrocyte apoptosis. Overexpression of c-Kit in an astrocyte-derived cell line in the absence of HIV-1 induces rapid apoptotic death. The apoptotic mechanism requires the c-Kit tyrosine kinase domain. The mechanism of c-kit induction by HIV-1 involves transactivation of the c-kit promoter by the HIV-1 Nef protein. These studies demonstrate that c-Kit can induce astrocyte apoptosis and suggest that this mechanism may play a role in CNS injury caused by HIV-1 infection. We propose that c-Kit can serve dual functions as a growth factor receptor or apoptosis inducer.

PubMed Disclaimer

Figures

Figure 1
Figure 1

Induction of c-kit by HIV-1 infection is associated with astrocyte apoptosis. Primary human fetal brain cultures (BC) were infected with HIV-189.6 or mock-infected, and c-Kit expression was examined by RT–PCR (a), Northern blot (b), and Western blot (c) analyses and immunofluorescence staining (d–f) at 30 days after infection. Endogenous c-Kit is detected in HEL and U87 cells but not in Jurkat, CEM, and 293 cells (b and c). (a) RT–PCR analysis of c-kit and β-actin transcripts. (b) Northern blot analysis of c-kit mRNA (5.5 kb) in total RNA (30 μg) isolated from mock- and HIV-1-infected brain cultures. The positions of 18S and 28S ribosomal RNA are indicated. (c) Western blot analysis of c-Kit (145 kDa). The two forms of c-Kit detected in U87 cells most likely correspond to the 120-kDa precursor and 145-kDa mature forms (25). (d–f) Double immunofluorescence staining of HIV-1-infected cultures with the primary antibodies indicated followed by fluorescein isothiocyanate- or rhodamine-secondary antibodies. (d) c-Kit colocalizes with the astrocyte-specific marker anti-glial fibrillary acidic protein. (e) Apoptosis in c-Kit-positive cells detected by TUNEL staining (arrows). (f) Apoptotic nuclear morphology in c-Kit-positive Nef-positive astrocyte (arrows) detected by nuclear staining with Hoechst 33342. (Left) Lower row is a double exposure. Cultures shown in f were treated with recombinant tumor necrosis factor α (10 ng/ml) for 48 h, which was necessary to permit detection of Nef in astrocytes (26). Treatment with tumor necrosis factor α did not change the expression of c-Kit or the relative number of apoptotic astrocytes in mock- versus HIV-1-infected cultures. Original magnifications: ×200 (d), ×300 (e), and ×400 (f). Results in a–f were similar in two or three experiments.

Figure 1
Figure 1

Induction of c-kit by HIV-1 infection is associated with astrocyte apoptosis. Primary human fetal brain cultures (BC) were infected with HIV-189.6 or mock-infected, and c-Kit expression was examined by RT–PCR (a), Northern blot (b), and Western blot (c) analyses and immunofluorescence staining (d–f) at 30 days after infection. Endogenous c-Kit is detected in HEL and U87 cells but not in Jurkat, CEM, and 293 cells (b and c). (a) RT–PCR analysis of c-kit and β-actin transcripts. (b) Northern blot analysis of c-kit mRNA (5.5 kb) in total RNA (30 μg) isolated from mock- and HIV-1-infected brain cultures. The positions of 18S and 28S ribosomal RNA are indicated. (c) Western blot analysis of c-Kit (145 kDa). The two forms of c-Kit detected in U87 cells most likely correspond to the 120-kDa precursor and 145-kDa mature forms (25). (d–f) Double immunofluorescence staining of HIV-1-infected cultures with the primary antibodies indicated followed by fluorescein isothiocyanate- or rhodamine-secondary antibodies. (d) c-Kit colocalizes with the astrocyte-specific marker anti-glial fibrillary acidic protein. (e) Apoptosis in c-Kit-positive cells detected by TUNEL staining (arrows). (f) Apoptotic nuclear morphology in c-Kit-positive Nef-positive astrocyte (arrows) detected by nuclear staining with Hoechst 33342. (Left) Lower row is a double exposure. Cultures shown in f were treated with recombinant tumor necrosis factor α (10 ng/ml) for 48 h, which was necessary to permit detection of Nef in astrocytes (26). Treatment with tumor necrosis factor α did not change the expression of c-Kit or the relative number of apoptotic astrocytes in mock- versus HIV-1-infected cultures. Original magnifications: ×200 (d), ×300 (e), and ×400 (f). Results in a–f were similar in two or three experiments.

Figure 2
Figure 2

Apoptosis induced by overexpression of c-Kit. U87 cells (5 × 105 cells) were transfected with 2.5 μg or the indicated amount of pcKit (○) or the pcDNA3 vector (•) by lipofection with N-[1-(2,3-dioleoyl)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP; Boehringer Mannheim). The transfection efficiency was 80–90% as determined by transfection with pCMV-βgal. (a) Cell survival was determined by counting the number of cells that excluded trypan blue. (b) The percentage of apoptotic cells was determined by analysis of nuclear morphology after staining with propidium iodide. (c and d) Analysis of DNA fragmentation on agarose gels. Soluble cytoplasmic DNA was isolated from cells transfected with pcKit at the indicated time points (c) or 48 h (d) after transfection. Apoptosis induced by staurosporine (0.1 mM for 16 h) gave the same pattern of DNA fragmentation as that shown in c and d, lanes 4–6 (data not shown). (e) Hoechst 33342 staining demonstrates apoptotic nuclei (arrows) in cells transfected with pcKit but not the control vector pCDNA3. (f) Apoptotic nuclei in c-Kit-positive cells (solid arrows) demonstrated by Hoechst 33342 staining. A normal nucleus is indicated by the open arrow. For e and f, cells were fixed at 48 h after transfection. Data in a and b are the means ± SD for duplicate determinations. Results shown are representative of at least two experiments.

Figure 3
Figure 3

Induction of apoptosis by c-Kit requires an intact tyrosine kinase domain. (a) Schematic diagram of c-Kit deletion mutants (ΔED, extracellular domain; ΔID, intracellular domain; ΔABM, ATP-binding motif ΔTKDI, tyrosine kinase domain I; ΔTKDII, tyrosine kinase domain II; ΔTKD, entire tyrosine kinase domain). (b) Expression of c-Kit mutants. Each plasmid at 20 μg was transfected into 293 cells by the calcium phosphate method. Immunoprecipitation of 500 μg of cellular protein followed by Western blotting with anti-c-Kit was performed at 48 h after transfection. (c and d) Induction of apoptosis by c-Kit mutants. Each plasmid at 2.5 μg was transfected into U87 cells as in Fig. 2. The cytoplasmic DNA fragmentation assay (c) and quantitation of apoptosis (d) were performed at 48 h after transfection as in Fig. 2 b–d. Data in d are the means ± SD for duplicate determinations. Results were similar in two or three experiments.

Figure 4
Figure 4

HIV-1 transactivation of the c-kit promoter. Transcriptional activation in 293 cells was determined by measuring CAT activity of the reporter construct pCD161 driven by the human c-kit promoter. 293 cells were transfected with 2 μg of CD161 and the indicated amounts (a–c) or 20 μg (d) of plasmid encoding full-length HIV-1 proviral DNA (NL4–3, SG3.1, YU2, or HXE) or HIV-1 Nef, Rev, Vpr, or Tat expression plasmids. Cell lysates prepared 48 h after transfection were assayed for CAT activity (20). All determinations were normalized to the activity of cotransfected pCMV-βgal. (a) pCD161 was cotransfected with different amounts of pNL4–3 HIV-1 proviral DNA. (b–d) Transactivation of the c-kit promoter by different HIV-1 isolates (b), HIV-1 proteins (c), or nef alleles (d). The Nef(Eli) plasmid was used in c. The relative activity above the basal level is shown at the top of each thin layer chromatography plate or bar graph. In d, the cpm were from converted acetylated chloramphenicol. Results shown are from single experiments that were repeated two or three times with similar results.

Similar articles

Cited by

References

    1. Price R W, Brew B, Sidtis J, Rosenblum M, Scheck A C, Cleary P. Science. 1988;239:586–591. - PubMed
    1. Gabuzda D H, Ho D D, de la Monte S M, Rota T R, Sobel R A. Ann Neurol. 1986;20:289–295. - PubMed
    1. Gartner S, Markovits P, Markovits D M, Kaplan M H, Gallo R C, Popovic M. Science. 1986;233:214–218. - PubMed
    1. Saito Y, Sharer L R, Epstein L G, Michaels J, Mintz M, Louder M, Golding K, Cvetkovich T A, Blumberg B M. Neurology. 1994;44:474–481. - PubMed
    1. Tornatore C, Chandra R, Berger J R, Major E O. Neurology. 1991;44:481–487. - PubMed

Publication types

MeSH terms

Substances