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. 2020 Feb 14;94(5):e01384-19.
doi: 10.1128/JVI.01384-19. Print 2020 Feb 14.

Kaposi's Sarcoma-Associated Herpesvirus Viral Interleukin-6 Signaling Upregulates Integrin β3 Levels and Is Dependent on STAT3

Ricardo Rivera-Soto #  1   2 , Nathan J Dissinger #  2 , Blossom Damania  3   2   4
Affiliations

Kaposi's Sarcoma-Associated Herpesvirus Viral Interleukin-6 Signaling Upregulates Integrin β3 Levels and Is Dependent on STAT3

Ricardo Rivera-Soto et al. J Virol. .

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of two B-cell lymphoproliferative diseases and Kaposi's sarcoma, an endothelial-cell-driven cancer. KSHV viral interleukin-6 (vIL-6) is a viral homolog of human IL-6 (hIL-6) that is expressed in KSHV-associated malignancies. Previous studies have shown that the expression of the integrin β3 (ITGB3) subunit is induced upon KSHV infection. Here we report that KSHV vIL-6 is able to induce the expression of ITGB3 and increase surface expression of the αVβ3 integrin heterodimer. We demonstrated using small interfering RNA (siRNA) depletion and inhibitor studies that KSHV vIL-6 can increase ITGB3 by inducing STAT3 signaling. Furthermore, we found that secreted vIL-6 is capable of inducing ITGB3 in endothelial cells in a paracrine manner. Importantly, the ability to induce ITGB3 in endothelial cells seems to be specific to vIL-6, as overexpression of hIL-6 alone did not affect levels of this integrin. Our lab and others have previously shown that vIL-6 can induce angiogenesis, and we investigated whether ITGB3 was involved in this process. We found that siRNA depletion of ITGB3 in vIL-6-expressing endothelial cells resulted in a decrease in adhesion to extracellular matrix proteins. Moreover, depletion of ITGB3 hindered the ability of vIL-6 to promote angiogenesis. In conclusion, we found that vIL-6 can singularly induce ITGB3 and that this induction is dependent on vIL-6 activation of the STAT3 signaling pathway.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of three human malignancies: multicentric Castleman's disease, primary effusion lymphoma, and Kaposi's sarcoma. Kaposi's sarcoma is a highly angiogenic tumor that arises from endothelial cells. It has been previously reported that KSHV infection of endothelial cells leads to an increase of integrin αVβ3, a molecule observed to be involved in the angiogenic process of several malignancies. Our data demonstrate that the KSHV protein viral interleukin-6 (vIL-6) can induce integrin β3 in an intracellular and paracrine manner. Furthermore, we showed that this induction is necessary for vIL-6-mediated cell adhesion and angiogenesis, suggesting a potential role of integrin β3 in KSHV pathogenesis and development of Kaposi's sarcoma.

Keywords: Kaposi’s sarcoma-associated herpesvirus; integrin β3; viral IL-6.

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Figures

FIG 1
FIG 1
HUVEC stably expressing vIL-6 have increased ITGB3 mRNA and protein levels. (A) Relative ITGB3 mRNA expression in stable HUVEC normalized to the expression levels in EV-HUVEC. (B) Integrin β3 protein expression in the total cell lysate of stable HUVEC. (C) (Top) Relative luciferase expression from a luciferase reporter under the control of an ITGB3-promoter transfected into HEK293T cells. (Bottom) Immunoblots for vIL-6 and actin from transfected HEK293T cells. (D) Integrin αV protein expression in the total cell lysate of stable HUVEC. (E) Surface expression of αVβ3 integrin in stable HUVEC was measured using flow cytometry. The gray histogram represents EV HUVEC, and the white histogram represents vIL-6 HUVEC. **, P < 0.01; ***, P < 0.001.
FIG 2
FIG 2
vIL-6 induces ITGB3 expression in a paracrine manner. (A and B) HUVEC were treated with conditioned medium from EV- or vIL-6-expressing HUVEC for 24 h, followed by the comparison of ITGB3 mRNA levels (A) and protein levels (B). (C and D) Similar experiments were conducted using conditioned medium from EV- and vIL-6-expressing BJABs. (E) Conditioned media were collected from EV- and vIL-6-HUVEC in the presence of nonspecific mouse IgG or mouse anti-vIL-6 IgG. This conditioned medium was then placed on HUVEC. After 24 h, lysates were collected, and immunoblotting was performed for actin and ITGB3. CM, conditioned medium; NS, nonspecific. *, P < 0.05; ***, P < 0.001.
FIG 3
FIG 3
vIL-6 from lytically replicating cells promotes the expression of ITGB3 in a paracrine manner. (A) iSLK.219 cells were reactivated with doxycycline for 24 h, and the levels of ITGB3, vIL-6, and actin were detected by immunoblotting. (B) Lysates from reactivated (24 h post-doxycycline treatment) iSLK.219 and vIL-6-expressing HUVEC were resolved by SDS-PAGE, and vIL-6 expression was detected by immunoblotting. (C) HUVEC were treated for 24 h with conditioned medium from latent or reactivated iSLK.219 (24 h post-doxycycline treatment). Immunoblotting was performed with HUVEC lysates and iSLK.219 conditioned medium. (D) Conditioned media were collected from TREx-Rta-BCBL-1 after 24 h of DMSO (latent) or doxycycline (reactivated [react.]) treatment. Conditioned medium was supplemented with nonspecific mouse IgG or mouse anti-vIL-6 IgG and placed on HUVEC. After 24 h, lysates were collected, and immunoblotting was performed. BCBL-1 cell conditioned medium was processed in the same manner.
FIG 4
FIG 4
JAK/STAT3 signaling is necessary for vIL-6-induced ITGB3. (A) Whole-cell lysates from EV- and vIL-6-HUVEC were immunoprecipitated with a FLAG (vIL-6) antibody. The eluates and inputs were resolved by SDS-PAGE, and immunoblotting was performed for the indicated proteins. (B) EV- and vIL-6-HUVEC were transfected with siRNAs for 72 h, and lysates were probed for the indicated proteins. (C) EV- and vIL-6-HUVEC were treated with the STAT3 inhibitor cryptotanshinone (0 or 20 μM) for 48 h. Lysates were then collected, and immunoblotting was performed for the indicated proteins. (D) EV- and vIL-6-HUVEC were transfected with siRNAs for 48 h, and lysates were probed for the same proteins as for panel C. (E) HUVEC were transfected with siRNAs against a nontargeting control or STAT3. Twenty-four hours posttransfection, cells were treated with conditioned medium from EV- or vIL-6-HUVEC and incubated for an additional 24 h before lysates were collected and used for immunoblotting. NTC, nontargeting control; H, HUVEC.
FIG 5
FIG 5
Human IL-6 is not a strong inducer of ITGB3 as is vIL-6 in HUVEC. (A) Immunoblots of total cell lysates from HUVEC expressing EV, vIL-6, or hIL-6 treated for 48 h in the presence of recombinant hIL-6 at the indicated concentrations. (B) Immunoblots of HUVEC treated with conditioned medium from EV-, vIL-6-, and hIL-6-expressing HUVEC and BJABs. (C) Similar to panel A, but EV- and vIL-6-HUVEC were grown for 24 h in the presence of recombinant hIL-6 (rhIL-6; 250 ng/ml), soluble IL-6Rα (sIL-6R; 250 ng/ml), or both.
FIG 6
FIG 6
ITGB3 aids in vIL-6-HUVEC adhesion to ECMs. (A) EV- and vIL-6-HUVEC were transfected for 48 h with nontargeting control or ITGB3 siRNAs, and cell lysates were collected for immunoblotting to confirm knockdown efficiency. (B) Cells treated as for panel A were stained with calcein-AM and plated for 30 min on wells precoated with fibronectin. Unattached cells were removed by gentle washes, and fluorescence was measured to quantify the relative amounts of cells adhered to the ECM component. (C) Similar to panel B, but with vitronectin-coated wells. Panels B and C represent the averages from three experiments, each with seven technical replicates. ***, P < 0.001.
FIG 7
FIG 7
ITGB3 contributes to vIL-6-induced tubule formation of endothelial cells. (A) Representative images from four experiments performed in duplicate with EV- and vIL-6-HUVEC treated with siRNAs against a nontargeting control or ITGB3. The center of the images was zoomed in for a better resolution of the tubules. (B) The average number of branching points per well (4 or 5 frames/well) in duplicates was calculated and represented in the scatterplot graph. **, P < 0.01.
FIG 8
FIG 8
Model of vIL-6 induction of ITGB3. Expression of vIL-6 augments JAK/STAT3 activation increasing the levels of ITGB3, which results in higher surface expression of the heterodimer αVβ3 integrin. This process promotes vIL-6-induced endothelial cell adhesion to the ECM components fibronectin and vitronectin and promotes tubule formation. The illustration was designed using BioRender.
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