Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Jul 5;428(2):112-20.
doi: 10.1016/j.virol.2012.03.013. Epub 2012 Apr 21.

The KSHV viral IL-6 homolog is sufficient to induce blood to lymphatic endothelial cell differentiation

Valerie A Morris  1 , Almira S Punjabi, Robert C Wells, Cristina J Wittkopp, Richard Vart, Michael Lagunoff
Affiliations

The KSHV viral IL-6 homolog is sufficient to induce blood to lymphatic endothelial cell differentiation

Valerie A Morris et al. Virology. .

Abstract

The predominant tumor cell of Kaposi's Sarcoma (KS) is the spindle cell, a cell of endothelial origin that expresses markers of lymphatic endothelium. In culture, Kaposi's Sarcoma-associated herpesvirus (KSHV) infection of blood endothelial cells drives expression of lymphatic endothelial cell specific markers, in a process that requires activation of the gp130 receptor and the JAK2/STAT3 and PI3K/AKT signaling pathways. While expression of each of the KSHV major latent genes in endothelial cells failed to increase expression of lymphatic markers, the viral homolog of human IL-6 (vIL-6) was sufficient for induction and requires the JAK2/STAT3 and PI3K/AKT pathways. Therefore, activation of gp130 and downstream signaling by vIL-6 is sufficient to drive blood to lymphatic endothelial cell differentiation. While sufficient, vIL-6 is not necessary for lymphatic reprogramming in the context of viral infection. This indicates that multiple viral genes are involved and suggests a central importance of this pathway to KSHV pathogenesis.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Transient expression of latent genes do not induce lymphatic differentiation. (A) Constructs expressing individual latent viral genes were transiently transfected into TIME cells. 48 h post transfection, cell lysates were harvested and subjected to immunoblot analysis with the indicated antibodies. In addition, stable pooled TIME cells expressing LANA or a retroviral vector control were similarly analyzed. While expression of individual latent viral genes was confirmed by FLAG or LANA immunoblot, no viral gene activated STAT3 or induced VEGFR-3 expression. (B) TIME cells (2 × 105) were transduced with lentiviral preps of the indicated KSHV latent genes or pCGSW, a control lentivirus that expresses GFP. 72 hpi, cell lysates were harvested and subjected to immunoblot analysis with the indicated antibodies. TIME cells were mock or KSHV-infected and harvested 48 hpi for positive and negative controls respectively.
Fig. 2
Fig. 2
Expression of vIL-6 but not other lytic genes that activate AKT induce lymphatic differentiation. (A) TIME cells expressing the lytic viral proteins vIL-6, vOX2, HA-K3, or FLAG-K5 were created by retroviral transduction and antibiotic selection. Cell lysates were assayed by immunoblot analysis with the indicated antibodies. Stable vIL-6 expression induced VEGFR-3 expression and activated STAT3. (B) TIME cells (2 × 105) were transiently transduced with lentiviral preps of the indicated KSHV lytic genes or a control lentivirus (pCGSW). 72 hpi, cell lysates were harvested and subjected to immunoblot analysis with the indicated antibodies. TIME cells were mock or KSHV-infected and harvested 48 hpi for positive and negative controls respectively.
Fig. 3
Fig. 3
Stable vIL-6 expression in TIME cells induces LEC-specific markers. (A) Stable vIL-6-expressing TIME cells induce VEGFR-3 and podoplanin expression compared to stable TIME cells expressing retroviral vector alone (pBMN-ZIN) as determined by immunoblot analysis. Mock and KSHV-infected TIME cells harvested 48 hpi were used as negative and positive controls. Stable vIL-6-expressing TIME cells also display persistent activation of STAT3 (B) Stable vIL-6-expressing TIME cells induce Prox1 and gp130 mRNA expression as determined by quantitative real-time RT-PCR. Error bars represent standard error of the mean (n=3).
Fig. 4
Fig. 4
vIL-6 reprograms primary blood endothelial cells to express lymphatic markers. Primary human blood microvascular endothelial cells (BECs) (2 × 105) were transiently transduced with lentiviral preps of vIL-6 lytic or control lentivirus (pCGSW). 72 hpi, cell lysates were harvested and subjected to immunoblot analysis with the indicated antibodies. Primary BECs were mock or KSHV-infected and harvested 48 hpi for positive and negative controls respectively.
Fig. 5
Fig. 5
vIL-6 induction of lymphatic differentiation requires JAK2/STAT3 and PI3K/AKT. (A) The PI3K inhibitor LY294002 (LY) and the JAK2 inhibitor AG490 (AG) block VEGFR-3 and podoplanin expression in stable vIL-6-expressing TIME cells. The numbers below the lanes indicated the relative abundance of VEGFR-3 and podoplanin expression normalized to β-actin expression in each lane compared to expression in the untreated vIL-6 expression cells. (B) Conditioned media harvested from stable vIL-6-expressing TIME cells transiently induces AKT and STAT3 phosphorylation in serum starved TIME cells treated for 30 min.
Fig. 6
Fig. 6
Knockdown of vIL-6 inhibits conditioned media from vIL-6 expression cells from inducing STAT3 but not from KSHV infected cells. (A) TIME cells were infected with KSHV, 24 hpi cells were transduced with lentiviruses encoding nonspecific shRNA (NS) or a vIL-6-specific shRNA. Cell lysates were harvest 48 h after transduction and subjected to immunoblot analysis with the indicated antibodies. Knockdown of vIL-6 had little or no effect on expression of lymphatic genes or STAT3 activation in infected cells. The numbers below the lanes indicated the relative abundance of phospho-STAT3 normalized to total STAT3 expression in each lane compared to expression in the NS control cells. (B) TIME cells were infected and transduced with vIL-6 specific or nonspecific shRNAs as in A. At 40 h post lentiviral transduction, the media was changed, conditioned media was harvested 8 h later and used to treat serum-starved TIME cells for 30 mins. As a control for vIL-6 knockdown, vIL-6 expressing TIME cells were concurrently infected with the shRNA lentiviruses and either pCGSW or vIL-6 expressing lentivirus. The vIL-6 shRNA decreased the phosphorylation of STAT3 by conditioned media harvested from control vIL-6 expressing cells (lanes 7 and 8), but not from KSHV-infected cell conditioned media (lanes 3 and 4). The numbers below the lanes indicated the relative abundance of phospho-STAT3 normalized to total STAT3 expression in each lane compared to expression in the NS control cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aluigi MG, Albini A, Carlone S, Repetto L, De Marchi R, Icardi A, Moro M, Noonan D, Benelli R. KSHV sequences in biopsies and cultured spindle cells of epidemic, iatrogenic and Mediterranean forms of Kaposi’s sarcoma. Res Virol. 1996;147:267–275. - PubMed
    1. Antman K, Chang Y. Kaposi’s sarcoma. N Engl J Med. 2000;342:1027–1038. - PubMed
    1. Aoki Y, Jaffe ES, Chang Y, Jones K, Teruya-Feldstein J, Moore PS, Tosato G. Angiogenesis and hematopoiesis induced by Kaposi’s sarcoma-associated herpesvirus-encoded interleukin-6. Blood. 1999;93:4034–4043. - PubMed
    1. Boshoff C, Schulz TF, Kennedy MM, Graham AK, Fisher C, Thomas A, McGee JO, Weiss RA, O’Leary JJ. Kaposi’s sarcoma-associated herpesvirus infects endothelial and spindle cells. Nat Med. 1995;1:1274–1278. - PubMed
    1. Brinkmann MM, Glenn M, Rainbow L, Kieser A, Henke-Gendo C, Schulz TF. Activation of mitogen-activated protein kinase and NF-kappaB pathways by a Kaposi’s sarcoma-associated herpesvirus K15 membrane protein. J Virol. 2003;77:9346–9358. - PMC - PubMed

Publication types

MeSH terms