Insulin-like growth factor II receptor-mediated intracellular retention of cathepsin B is essential for transformation of endothelial cells by Kaposi's sarcoma-associated herpesvirus - PubMed (original) (raw)

Insulin-like growth factor II receptor-mediated intracellular retention of cathepsin B is essential for transformation of endothelial cells by Kaposi's sarcoma-associated herpesvirus

Patrick P Rose et al. J Virol. 2007 Aug.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the pathological agent of Kaposi's sarcoma (KS), a tumor characterized by aberrant proliferation of endothelial-cell-derived spindle cells. Since in many cancers tumorigenesis is associated with an increase in the activity of the cathepsin family, we studied the role of cathepsins in KS using an in vitro model of KSHV-mediated endothelial cell transformation. Small-molecule inhibitors and small interfering RNA (siRNA) targeting CTSB, but not other cathepsins, inhibited KSHV-induced postconfluent proliferation and the formation of spindle cells and foci of dermal microvascular endothelial cells. Interestingly, neither CTSB mRNA nor CTSB protein levels were induced in endothelial cells latently infected with KSHV. Secretion of CTSB was strongly diminished upon KSHV infection. Increased targeting of CTSB to endosomes was caused by the induction by KSHV of the expression of insulin-like growth factor-II receptor (IGF-IIR), a mannose-6-phosphate receptor (M6PR) that binds to cathepsins. Inhibition of IGF-IIR/M6PR expression by siRNA released CTSB for secretion. In contrast to the increased cathepsin secretion observed in most other tumors, viral inhibition of CTSB secretion via induction of an M6PR is crucial for the transformation of endothelial cells.

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Figures

FIG. 1.

Cysteine protease inhibitors effectively prevent KSHV-induced formation of spindle cell-containing foci. (A) Phase-contrast images of KSHV-infected E-DMVEC treated with cysteine protease inhibitor EST or LHVS. Both compounds effectively inhibit spindle cell and focus formation. (B) Western blot analysis comparing levels of protein expression for cathepsins S, L, and B in KSHV-infected and uninfected E-DMVEC. To control for equal loading, antibodies to the cellular chaperone calreticulin were used.

FIG. 2.

Gene expression profiling of cathepsin genes in KSHV-infected and uninfected E-DMVEC. (A) Affymetrix HG_U133A GeneChip data for cathepsins were obtained from four infected samples and compared to their respective controls. Asterisks indicate significant changes (P > 0.01 by analysis of variance). Genes with multiple probe sets are reflected in the table. NC, no change in gene expression; A, the probe set scored absent on the microarray; I, changes where gene expression was induced.

FIG. 3.

Downregulation of CTSB mRNA inhibits KSHV-mediated focus formation. (A) Western blot (WB) analysis of cathepsin S, L, and B protein levels after siRNA treatment. An antibody to calreticulin (α-CRT) was used to control for equal loading. siRNAs to cathepsins L and B were target specific as determined by Western blotting. (B) Representative phase-contrast microscopy comparing untreated samples to cathepsin S, L, or B siRNA-treated, KSHV-infected E-DMVEC. Control siRNA-treated samples were indistinguishable from untreated samples and are not represented here. Only CTSB siRNA reduced focus formation. Immunofluorescence analysis of Cy3-labeled siRNA transfectants indicated a transfection efficiency of >95% (data not shown).

FIG. 4.

Intracellular CTSB is essential for KSHV-induced spindle cell and focus formation. (A) Phase-contrast images of CTSB siRNA-treated cultures incubated with purified human native CTSB. Treatment of KSHV-infected E-DMVEC with 10 ng/ml native CTSB (rescue) did not reverse the effect of CTSB siRNA knockdown. The upper left panel shows KSHV-infected E-DMVEC without treatment. (B) Zymogen assay to determine the effectiveness of the two inhibitors of CTSB enzymatic activity. Analysis by cleavage of the CTSB fluorogenic substrate AMC showed that both inhibitors effectively reduced CTSB activity. (C) Representative phase-contrast images of KSHV-infected E-DMVEC that were either left untreated (panel 1) or treated with CA-074ME (panel 3) or CA-074 (panel 2). Only the membrane-permeant version of the CTSB inhibitor, CA-074ME, was able to inhibit spindle cell and focus formation in KSHV-infected E-DMVEC (panel 2). (D) Microscopy images of the fluorescently tagged cathepsin L and B activity probe GB-111 FL after treatment with a competitive CTSB inhibitor or a noncompetitive insulin receptor inhibitor [HNMPA-(AM3)]. KSHV-infected E-DMVEC were treated with the various inhibitors overnight before being probed with the activity-based small-molecule inhibitor GB-111 FL. Only CA-074ME treatment dramatically reduced the staining pattern of GB-111 FL (panel 4). Untreated (panel 1), HNMPA-(AM3)-treated (panel 2), and CA-074-treated (panel 3) cells showed no change in their staining. Some cells treated with CA-074 did show a reduction in vesicular staining similar to that with CA-074ME, and it has been documented that CA-074 can permeate the cells; however, at this concentration the inhibitor had no effect on KSHV-induced transformation of E-DMVEC.

FIG. 5.

The membrane-permeant CTSB inhibitor CA-074ME reduces the number of LANA-1-positive E-DMVEC. (A) Phase-contrast and dark-field images of untreated (top), CA-074ME-treated (10 μM) (center), and CA-074-treated (10 μM) (bottom) KSHV-infected E-DMVEC showing expression of LANA-1 in KSHV-infected spindle cells and the selective loss of 89% of LANA-1-positive foci following CA-074ME treatment. (B) Graph quantifying LANA-1-positive cells for each field visualized.

FIG. 6.

CTSB inhibitors are nontoxic and do not induce apoptosis. (A) ATP cell viability assay. KSHV-infected E-DMVEC were either left untreated or treated with either 10 μM CA-074, 10 μM CA-074ME, or 100 μM HNMPA-(AM3). After treatment, cells were lysed, and ATP was quantitatively determined using a luciferin bioluminescence assay. Neither CA-074 nor CA-074ME was toxic to the cells. (B) Annexin V-FITC apoptosis detection assay. Uninfected (panels 1 to 4) and KSHV-infected (panels 5 to 8) E-DMVEC were double-stained with propidium iodide and Annexin V-FITC. No significant difference in apoptosis was seen between untreated cells (panels 1 and 5) and cells treated with 10 μM CA-074 (panels 3 and 7) or CA-074ME (panels 4 and 8). Staurosporine was used as a positive control (panels 2 and 6). Lower left quadrant, healthy cells, lower right, early-apoptotic cells; upper right, late-apoptotic cells.

FIG. 7.

Inhibiting intracellular CTSB activity prevents postconfluent anchorage-independent growth of KSHV-infected E-DMVEC. (A) Preconfluent cell proliferation. Cells were seeded at a low density and were either left untreated or treated with either CA-074, CA-074ME, or HNMPA-(AM3). Total-cell counts were determined using a hemocytometer and were compared to normal cell proliferation of untreated E-DMVEC. (B) Postconfluent cell proliferation. Uninfected or KSHV-infected E-DMVEC were seeded at a high density and treated with either CA-074, CA-074ME, or HNMPA-(AM3). Total-cell numbers of KSHV-infected E-DMVEC, counted using a hemocytometer, are presented as a percentage of uninfected E-DMVEC total-cell counts in order to determine differences based on any effect of CA-074, CA-074ME, or HNMPA-(AM3) treatment. (C) Soft-agar growth assay. KSHV-infected E-DMVEC were either left untreated or treated with either CA-074 or CA-074ME for 14 days and transferred to a soft-agar-containing medium. Phase-contrast images are representative. (D) Graph quantifies the number of foci observed in each sample; data are means from two replicate experiments.

FIG. 8.

Active CTSB is retained in KSHV-infected E-DMVEC, and both uninfected and KSHV-infected E-DMVEC express the same CTSB mRNA species. (A) Zymogen assay comparing CTSB enzymatic activities of uninfected and KSHV-infected E-DMVEC cultures in lysates and supernatants. Uninfected E-DMVEC secrete the majority of active CTSB, while KSHV-infected E-DMVEC retain almost all of the active CTSB intracellularly, as very little is secreted. (B) RT-PCR determining CTSB mRNA species in uninfected and KSHV-infected E-DMVEC. The hypoxanthine phosphoribosyltransferase (HPRT) housekeeping gene was used as a positive control. Both uninfected and KSHV-infected cells have truncated versions of CTSB mRNA as the dominant species; they are missing either exon 2 or exons 2 and 3.

FIG. 9.

IGF-IIR/M6PR is responsible for increased endolysosomal trafficking of CTSB. (A) CTSB activity assay after disruption of lysosomes with NH4Cl. Uninfected and KSHV-infected E-DMVEC lysates and supernatants were collected 24 h after NH4Cl treatment, and CTSB activity was compared to that for untreated samples. NH4Cl efficiently disrupted intracellular CTSB activity. (B) CTSB zymogen assay controlling for activity in the presence of NH4Cl. NH4Cl had no effect on actual CTSB activity. (C) Quantitative real-time PCR comparing IGF-IR and IGF-IIR/M6PR expression levels in KS tissue. Only IGF-IIR/M6PR was significantly induced in KS tissue. (D) Coimmunoprecipitation of IGF-IIR/M6PR and CTSB. Lysates of uninfected and KSHV-infected E-DMVEC were lysed and immunoprecipitated (IP) with anti-IGF-IIR/M6PR. Samples were resolved on a 10% acrylamide gel and blotted with an anti-CTSB antibody. There was an increase in the level of coimmunoprecipitated CTSB proportional to the increase in IGF-IIR/M6PR expression after long-term KSHV infection. WB, Western blotting. (E) Quantitative real-time PCR confirming efficient knockdown of IGF-IIR/M6PR mRNA after siRNA treatment against IGF-IIR/M6PR. The siRNA had no effect on IGF-IR. (F) Representative assay of CTSB activity after 1 week of IGF-IIR/M6PR siRNA treatment. Cells were treated again using the two-hit combination 1 day prior to testing of CTSB activity. In the presence of IGF-IIR/M6PR siRNA, intracellular CTSB activity was depleted, indicating that IGF-IIR/M6PR is responsible for the increased endolysosomal targeting of CTSB.

FIG. 10.

IGF-IIR/M6PR siRNA inhibits spindle cell and focus formation in KSHV-infected E-DMVEC. Shown are representative phase-contrast microscopy images comparing untreated samples to IGF-IIR/M6PR siRNA-treated, KSHV-infected E-DMVEC. Control siRNA-treated samples were indistinguishable from untreated samples and are not represented here. IGF-IIR/M6PR siRNA reduced focus formation. Immunofluorescence analysis of Cy3-labeled siRNA transfectants indicated a transfection efficiency of >95% (data not shown).

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Insulin-like growth factor II receptor-mediated intracellular retention of cathepsin B is essential for transformation of endothelial cells by Kaposi's sarcoma-associated herpesvirus - PubMed (original) (raw)