KSHV-induced notch components render endothelial and mural cell characteristics and cell survival

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV) infection is essential to the development of Kaposi sarcoma (KS). Notch signaling is also known to play a pivotal role in KS cell survival and lytic phase entrance of KSHV. In the current study, we sought to determine whether KSHV regulates Notch components. KSHV-infected lymphatic endothelial cells showed induction of receptors Notch3 and Notch4, Notch ligands Dll4 and Jagged1, and activated Notch receptors in contrast to uninfected lymphatic endothelial cells. In addition, KSHV induced the expression of endothelial precursor cell marker (CD133) and mural cell markers (calponin, desmin, and smooth muscle alpha actin), suggesting dedifferentiation and trans-differentiation. Overexpression of latency proteins (LANA, vFLIP) and lytic phase proteins (RTA, vGPCR, viral interleukin-6) further supported the direct regulatory capacity of KSHV viral proteins to induce Notch receptors (Notch2, Notch3), ligands (Dll1, Dll4, Jagged1), downstream targets (Hey, Hes), and endothelial precursor CD133. Targeting Notch pathway with gamma-secretase inhibitor and a decoy protein in the form of soluble Dll4 inhibited growth of KSHV-transformed endothelial cell line. Soluble Dll4 was also highly active in vivo against KS tumor xenograft. It inhibited tumor cell growth, induced tumor cell death, and reduced vessel perfusion. Soluble Dll4 is thus a candidate for clinical investigation.

Figure 1

Expression of Notch pathway genes in KS cells. (A) Expression of Notch receptors (top panel), Notch ligands (middle panel), and Notch pathway downstream target genes (bottom panel) in endothelial cells and KS cells was analyzed by RT-PCR. HUAECs, HUVECs, and LECs were included as controls. KSHV-infected LECs (LEC/KSHV) were treated with TPA (25 ng/mL) or dimethyl sulfoxide (DMSO) for 48 hours before harvest. KS-IMM was a tumor cell line isolated from KS lesion. β-Actin expression indicates that an equal amount of RNA was used to perform PCR. (B) Expression of Notch3 in human KS tumor tissue was analyzed by immunostaining. The KS tumor regions are indicated by arrows. (C) Expression of Notch receptors and Notch ligands in KS cells were analyzed by Western blot. The relative expression level was quantitated by ImageJ (National Institutes of Health), normalized to β-actin, and is shown below each panel. (D) sDll4-Fc or human Fc fragment was clustered with anti-Fc antibody (1:1 ratio) at room temperature for 60 minutes. LEC/KSHV cells were then treated with clustered sDll4-Fc or Fc fragment (1 μg/mL) for 90 minutes, and whole cell lysate was subjected to Western blot with antibody against activated Notch.

Figure 2

KS cells have both endothelial and mural cell characteristics. (A) Expression of endothelial progenitor markers (CD133 and CD248) in KS cells was analyzed by RT-PCR. (B) LECs and LEC/KSHV cells were sorted for cell surface CD133 expression by flow cytometry. (C) Expression of mural cell markers (calponin, desmin, and α-SMA) in KS cells was analyzed by RT-PCR. Human UASMCs were used as positive control for mural cell markers. (D) Western blots analysis for α-SMA expression in KS cells. (E) Cells were labeled with green or orange CellTracker and cultured on Matrigel for 6 hours, alone or in various combinations. (Right panel) Enlarged boxed area in the middle panel. Arrows point to the tubes formed by integration of 2 types of endothelial cells. Arrowheads point to the endothelial cells lined with mural cells.

Figure 3

Induction of Notch pathway genes by KSHV proteins. The 293T cells were transfected with expression vectors for latency phase genes (LANA or vFLIP; A) and lytic phase genes (RTA, vGPCR, or vIL-6; B). Empty pcDNA3 vector transfection was used as control. Expression of Notch receptors (left panel), Notch ligands (middle panel), and Notch pathway downstream genes (right panel) was analyzed by quantitative RT-PCR 72 hours after transfection. (C) The overall change of expression of Notch pathway genes after transfection was summarized. − indicates without significant induction; and +, with significant induction. (D) Expression of endothelial progenitor marker CD133 was analyzed by quantitative RT-PCR as in panels A and B.

Figure 4

vGPCR and vFLIP proteins regulate Notch pathway through ERK and NF-κB, respectively. (A) HUVECs stably transfected with vFLIP-ER^TAM plasmid or control vector were grown on 6-well plates and induced by 4-OHT (100nM) for 48 hours. The cells were treated with DMSO or Bay (10μM) for 2 hours. Cells without 4-OHT induction were also treated with Bay (10μM) for 2 hours before harvest. RNA was isolated and subjected to quantitative RT-PCR. Expression of Dll4, Jagged1, and Hey1 was analyzed. (B) Cells were treated in the same way as in panel A and subjected to Western blot for Dll4 and Jagged1. (C) 293T cells were transfected with vGPCR plasmid for 72 hours. Before harvest, the vGPCR-transfected cells were treated by PI3K inhibitor LY (LY294002, 15μM), ERK inhibitor U0126 (15μM), and NF-κB inhibitor Bay (Bay 11-7085, 10μM), or a combination of the 3. Cells were treated by LY and U0126 for 5 hours and Bay for 2 hours. RNA was isolated and subjected to quantitative RT-PCR. Expression of Dll4, Jagged1, and Hey1 was analyzed.

Figure 5

Notch signaling in KS cell survival and mural cell characteristic. (A) LEC, LEC/KSHV, and KS-IMM cells were treated by sDll4-Fc (4 μg/mL) or GSI-1 (1μM) for 48 hours. Purified human Fc fragment (4 μg/mL) and DMSO were used as control for sDll4-Fc and GSI-1, respectively. Cell viability was analyzed by MTT assay and normalized to control. (B) Cells were treated in the same way as in panel A but were harvested and subjected to quantitative RT-PCR. Expression of Notch pathway downstream genes was analyzed. (C) LEC/KSHV cells were transfected by Notch3 siRNA (20nM or 100nM) for 72 hours. Nonsilencing siRNA from QIAGEN (100 nM) was used as negative control. Cells were harvested, and Notch3 RNA level change after siRNA transfection was analyzed by quantitative RT-PCR. Expression level changes of putative Notch3 downstream genes were also analyzed. (D) Cells were treated in the way same as in panel C and subjected to Western blotting for Notch3 and α-SMA. The relative protein level of Notch3 and α-SMA was quantitated by ImageJ and normalized to β-actin level (E) Cells were treated as in panel C and subjected to MTT assay. Cell viability was normalized to mock transfection.

Figure 6

sDll4-Fc inhibits KS tumor growth in vivo. (A) Athymic mice were implanted with 2 × 10⁶ KS-IMM cells. When tumor sizes were approximately 100 mm³, mice were randomly assigned to treatment groups (6 per group) (day 0). Mice were treated by intraperitoneal injection of sDll4-Fc (10 mg/kg) or PBS, 3 times a week for 31 days. Tumor volume was measured 3 times a week. The P value was calculated by Student t test. (B) Just before tissue harvest, mice were infused with RCA-Lectin and hypoxyprobe. Tumor tissue structure was examined by hematoxylin and eosin staining. Perfused vessels were localized by RCA-Lectin, microvascular endothelial cells were localized by CD31 staining, and pericytes were localized by NG2 staining. Nuclei were counterstained with 6-diamidino-2-phenylindole dihydrochloride. Proliferating cells within the tumor were assessed by immunostaining with anti-Ki67 antibody. Apoptosis was examined with TUNEL assay. Hypoxia was assessed by immunostaining with anti-Hypoxyprobe antibody. Quantitation was performed with the use of Bioquant Image Analysis (Bioquant), and the relative fluorescence level is shown below the panel. The confocal images were taken at an original magnification ×60.