Virus Genes

, Volume 36, Issue 1, pp 147–155

Signal transduction pathways utilized by enzootic nasal tumor virus (ENTV-1) envelope protein in transformation of rat epithelial cells resemble those used by jaagsiekte sheep retrovirus

Authors

  • Naoyoshi Maeda
    • Cancer Research InstituteUniversity of California, Irvine
    • Department of Molecular Biology and BiochemistryUniversity of California, Irvine
    • Division of Host Defense, Research Center for Prevention of Infectious Diseases, Medical Institute of BioregulationKyushu University
    • Division of Bioinformatics, Digital Medicine InitiativeKyushu University
    • Cancer Research InstituteUniversity of California, Irvine
    • Department of Molecular Biology and BiochemistryUniversity of California, Irvine
Article

DOI: 10.1007/s11262-007-0193-x

Cite this article as:
Maeda, N. & Fan, H. Virus Genes (2008) 36: 147. doi:10.1007/s11262-007-0193-x
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Abstract

The ovine β-retroviruses enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV) are the causative agent of enzootic nasal adenocarcinoma (ENA) and ovine pulmonary adenocarcinoma (OPA), respectively, characterized by neoplastic transformation of secretory epithelial cells. The Envelope (Env) proteins of these related betaretroviruses act as oncogenes, in that they can transform fibroblast and epithelial cell lines in culture. In addition, viral vector-mediated expression of the Env proteins for these viruses causes tumors in animals. Here, we investigated what signaling pathways are required for the ENTV transformation in vitro. We have previously found that Ras–MEK–MAPK and PI3k–Akt–mTOR are involved in JSRV transformation of fibroblast and epithelial cells. In this study, we found that the MEK inhibitor PD98059 and mTOR inhibitor Rapamycin inhibited ENTV transformation in RK3E rat kidney epithelial cells, but the p38 inhibitor SB203580 drastically enhanced transformation, which is quite similar to JSRV transformation. Small molecular inhibitors and dominant negative versions of H-ras and Rac1 indicated a role for both of these molecules in transformation by either virus. These results indicate that the signaling pathways for ENTV and JSRV transformation are quite similar, consistent with the notion that these proteins do not determine the tissue-specificity of the tumors for these viruses.

Keywords

RetrovirusENTVJSRVEnvelopeTransformationSignal transduction

Introduction

The ovine β-retroviruses Enzootic Nasal Tumor Virus enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV) are the causative agent of enzootic nasal adenocarcinoma (ENA) and ovine pulmonary adenocarcinoma (OPA) in nasal glands and the lungs, respectively [1, 2]. JSRV infection in sheep with naturally occurring OPA was previously reported [3]. These viruses are closely related to each other and to endogenous sheep retroviruses (ESRVs) at nucleotide sequence level. The sequence of the complete genome of ENTV derived from sheep (ENTV-1) was determined [4]. Recently another sequence of a new isolate derived from goats (ENTV-2) was also determined [5]. These isolates are currently regarded as distinct viruses. Specific PCR revealed ENTV-2 establishes a disseminated lymphoid infection, while ENTV-1 is mainly confined to the tumor [5].

Investigators have revealed that JSRV Envelope (Env) functions as an oncoprotein, since it can transform in vitro several fibroblast (rodent NIH-3T3, Rat6, and 208F or chicken DF-1) [614] and epithelial cell lines (human BEAS-2B, canine MDCK or rat RK3E and IEC-18) [1518]. In addition, viral vectors expressing only JSRV Env induced lung tumors in sheep [19] and mice [2022], suggesting JSRV Env itself is necessary and sufficient to induce lung tumors in vivo. ENTV-1 Env also has been shown to transform rodent fibroblast and epithelial cell lines [6, 8, 9, 16, 23], and to induce lung tumors in mice [21].

The mechanisms of transformation of epithelial cells by JSRV Env were studied by several laboratories. Retroviral Env proteins are translated as a single polyprotein precursor that is subsequently cleaved into the SU (surface) and TM (transmembrane) proteins. A variety of experiments on JSRV indicated that the short 45 amino acid cytoplasmic tail of the TM protein is crucial for transformation [6, 7, 9, 11, 12, 14, 1618, 24]. Presumably residues in the cytoplasmic tail contact with cytoplasmic cellular proteins, leading to activation of signaling cascades and cell transformation. For JSRV, we and other investigators found that Ras–MEK–MAPK and PI3k–Akt–mTOR signaling pathways are involved in JSRV transformation of epithelial cells in vitro [1518]. In addition, MAPK activation (phosphorylation of p44/42) was observed in experimental and natural OPA in vivo [17], and also in tumors induced by expression of JSRV envelope Env only [19].

Comparison of the transformation mechanisms by the ENTV-1 and JSRV Env is of great interest, since ENA arises from infected secretory epithelial cells of the ethmoid turbinate of the nasal cavity, while OPA is derived from secretory epithelial cells of the distal airways in the lungs (type II pneumocytes and Clara cells) [1, 2]. Conceivably this difference could reflect different cell-specific capacities for transformation by ENTV-1 vs. JSRV Env proteins. Indeed, the cytoplasmic tails of ENTV-1 and JSRV TM proteins show substantial amino acid variation [25]. However, Wootton et al. reported that infection of immunodeficient mice with an AAV6 vector expressing ENTV-1 Env resulted in lung tumors resembling OPA rather than tumors in the nasal cavity [21]. Moreover, immunohistochemical staining of ENA and OPA tissues revealed that Raf–MEK–MAPK activation was detected in both tumor types [26]. Thus the mechanisms of ENA and OPA envelope Env transformation might be similar.

In this study, we investigated the roles of different signaling pathways in ENTV-1 transformation in comparison with JSRV transformation in vitro. In addition, the experiments identified another signaling protein involved in transformation by these two viruses, Rac1.

Materials and methods

Constructs

The constructs for JSRV and ENTV-1 Env used in this study are described in Fig. 1. The JSRV Env expression plasmid pCMV3ΔGP(JSRV) and its Flag-tagged version pCMV3ΔGP(JSRV–FLAG) were described before [17]. The ENTV-1 Env expression plasmid pCMV3ΔGP(ENTV) [6] was provided by Dr. Massimo Palmarini. pCMV3ΔGP(ENTV–FLAG) was made by inserting the Flag-tag at the C-terminus of ENTV-1 env open-reading frame. The sequencing was performed by the UCI DNA sequencing core facility. Raf-CAAX is a human Raf-1 kinase fused at the C-terminus with a plasma membrane-targeting sequence (18 amino acids) of K-Ras4B [27, 28]. H-ras(17N) [29] and Rac1(17N) [30, 31] express dominant negative mutants of H-ras and Rac1, respectively. Raf-CAAX, H-ras(17N) and Rac1(17N) were provided by Dr. Eric Stanbridge.
https://static-content.springer.com/image/art%3A10.1007%2Fs11262-007-0193-x/MediaObjects/11262_2007_193_Fig1_HTML.gif
Fig. 1

JSRV and ENTV constructs used. The plasmids are detailed in Materials and Methods. pCMV3ΔGP(JSRV) expresses the JSRV Env from a CMV promoter, and pCMV3ΔGP(JSRV–FLAG) was described before. The backbone of pCMV3ΔGP(ENTV) is pCMV3ΔGP(JSRV), and JSRV Env was replaced with ENTV Env. The FLAG-tag (closed box) are inserted in wild-type pCMV3ΔGP(ENTV) at the C-terminus of the env reading frame the same way that pCMV3ΔGP(JSRV–FLAG) was made. SD; env splice donor, SA; splice acceptor

Cells

Rat kidney epithelia RK3E cells and human embryonic kidney epithelia 293T cells were grown in Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin (100 μg/ml).

Inhibitors

The pharmacological inhibitors, FTI-277, NSC 23766, PD98059, SB203580, and rapamycin (CALBIOCHEM) were added at every medium change at the concentrations indicated.

Transfection

RK3E cells or 293T cells were seeded at 5 × 105 cells/plate in 6 cm dishes 24 h before transfection. Five micrograms of plasmid DNA were transfected to RK3E cells with FuGENE 6 Transfection Reagent (Roche). For transient gene expression, the transfected 293T cells were cultured for 48 h and harvested for western blot analysis. For transformation assays, the transfected RK3E cells were cultured for up to four weeks, and the number of transformed foci were counted under phase contrast microscopy. When large numbers of foci were present, the colonies present in one quarter of the dish were counted and the values were multiplied by four.

Immunoblotting

Cells were lysed with Triton Lysis buffer (50 mM Tris–Cl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 10% Glycerol, and Phosphatase Inhibitor Cocktail 2 (Sigma)) for 30 min. Protein samples (10–30 μg per sample) were mixed with SDS sample buffer (0.35 M Tris–HCl (pH 6.8), 10.28% (w/v) SDS, 36% (v/v) glycerol, 5% β-mercaptoethanol, 0.012% (w/v) bromophenol blue) followed by boiling for 5 min., and eventually subjected to SDS-PAGE and immunoblot analysis. Monoclonal or polyclonal antibodies against Flag-tag (Sigma) were used as primary antibodies. Secondary antibodies appropriate for the species of the primary antibodies were then used. Goat anti-mouse and goat anti-rabbit (Pierce) IgG-conjugated with horseradish peroxidase were used as secondary antibodies. Blots were visualized by the SuperSignal West Pico Chemiluminescent Substrate (Pierce).

Results

ENTV-1 Env protein transforms RK3E rat kidney epithelial cells

Initial studies demonstrating the transforming potential of JSRV and ENTV-1 Env proteins were performed in NIH-3T3 mouse fibroblast and Rat-6 or 208F rat fibroblast cell lines [6, 814, 23]. Subsequently we and others have focused on transformation of epithelial cell lines [1518], since the target cells for JSRV and ENTV-1 oncogenesis are secretory epithelial cells (lung type II pneumocytes and Clara cells for JSRV and ethmoid tubinate cells for ENTV-1) [1, 2]. We have previously reported that JSRV Env protein transforms RK3E rat kidney epithelial cells [17], and we tested if ENTV ENTV-1 Env can transform these cells. We transfected RK3E cells with CMV promoter-driven expression plasmids for JSRV or ENTV-1 Env (pCMV3ΔGP(JSRV) and pCMV3ΔGP(ENTV) respectively; Fig. 1) as well as with FLAG epitope-tagged versions. The transfected cultures were then incubated under assay conditions for transformed cell focus formation (Fig. 2). As a positive control, RK3E cells were also transfected with an expression plasmid for a constitutively active human Raf proto-oncogene. As expected, pCMV3ΔGP(ENTV) induced foci of transformation with similar characteristics to pCMV3ΔGP(JSRV)-induced foci or activated Raf-induced foci. Quantification in Table 1 indicated that ENTV-1 Env transformed RK3E cells with equivalent (or slightly higher) efficiency than JSRV Env. Addition of the C-terminal Flag epitope somewhat reduced the efficiency of ENTV-1 Env transformation, as previously observed for JSRV Env [17].
https://static-content.springer.com/image/art%3A10.1007%2Fs11262-007-0193-x/MediaObjects/11262_2007_193_Fig2_HTML.gif
Fig. 2

Transformation of immortalized rat kidney epithelial cells, RK3E. RK3E cells were transfected with 5 μg of (a) pcDNA3.1(-) (negative control), (b) Raf-CAAX, (c) pCMV3ΔGP(JSRV), or (d) pCMV3ΔGP(ENTV). Foci of transformed cells at day 17 after transfection are shown. Magnification 100x in all panels

Table 1

Focus formation in RK3E cellsa

 

Experiment 1

Experiment 2

pcDNA3.1(-)

0

0

pCMV3ΔGP(JSRV)

1256

924

pCMV3ΔGP(ENTV)

1624

1052

pCMV3ΔGP(JSRV–FLAG)

854

564

pCMV3ΔGP(ENTV–FLAG)

896

604

aRK3E cells were transfected with the 5 μg plasmid DNAs in 6 cm dish as described in Materials and Methods, and the numbers of transformed foci were scored at day 17 after transfection

Effects of MEK, p38 and Akt inhibitors on ENTV-1 transformation

We and others have previously investigated the signal transduction pathways involved in JSRV transformation in fibroblast and epithelial cell lines [618, 23, 24]. So far, the PI3K–Akt–mTOR and Ras–MEK–MAPK pathways have been found to be involved. In order to investigate the roles of these pathways in ENTV-1 transformation, we used pharmacological inhibitors of mTOR (rapamycin), MEK-1 (PD98059), and p38 MAPK (SB203580). Similar to JSRV transformation of RK3E, ENTV-1 transformation was almost completely inhibited by PD98059, indicating an essential role for signaling through MEK-1 for transformation (Table 2). ENTV-1 transformation was also quite strongly inhibited by rapamycin (ca. 88%), indicating an important role for signaling through mTOR for transformation as well. The inhibition by rapamycin was stronger for ENTV-1 transformation than JSRV transformation. We previously showed that inhibition of p38 with SB203580 strongly enhanced the efficiency of JSRV transformation in RK3E and NIH-3T3 cells, indicating that p38 negatively regulates JSRV transformation. Similarly, transformation by ENTV-1 Env was also enhanced by SB203580 (Table 2).
Table 2

Effects of inhibitors on transformation of RK3E cellsa

 

Experiment 1

Experiment 2

Inhibition (%)

PD98059 (μM)

0

10

0

10

 

pcDNA3.1(-)

0

0

0

0

 

pCMV3ΔGP(JSRV)

1112

0

1008

0

100

pCMV3ΔGP(ENTV)

1744

28

1424

16

98.6 ± 0.3

 

Experiment 1

Experiment 2

Enhancement

SB203580 (μM)

0

3

0

3

 

pcDNA3.1(-)

0

0

0

0

 

pCMV3ΔGP(JSRV)

416

2860

372

2584

6.9 ± 0.1 X

pCMV3ΔGP(ENTV)

460

3924

484

4092

8.5 ± 0.1 X

 

Experiment 1

Experiment 2

Inhibition (%)

Rapamycin (ng/ml)

0

5

0

5

 

pcDNA3.1(-)

0

0

0

0

 

pCMV3ΔGP(JSRV)

1112

264

1284

332

75.2 ± 1.1

pCMV3ΔGP(ENTV)

1744

184

1668

228

87.9 ± 1.6

aRK3E cells were transfected with the 5 μg plasmid DNAs in 6 cm dish as described in Materials and Methods, and the numbers of transformed foci were scored at day 17 (PD98059 and rapamycin) and day 11 (SB203580) after transfection. Inhibitors were added daily at the concentrations indicated

We isolated two transformed clones of RK3E cells transfected with the FLAG-tagged ENTV-1 Env protein and confirmed that they were expressing Env protein (uncleaved Env polyprotein as well as the cleaved TM protein) by western blot analysis for the FLAG epitope (not shown). Treatment of the two ENTV-1-transformed cell clones with PD98059 and SB203580 is shown in Fig. 3, in comparison to a JSRV-transformed RK3E clone. Inhibition of MEK-1 by PD98059 resulted in partial reversion of the transformed morphology (clone 1, panel h vs. g) or reduction in growth rate (clone 2, panel k vs. j), similar to the effects on the JSRV-transformed clone (panel e vs. d). Inhibition of p38 with SB203580 increased the growth rates of both ENTV-1 transformant clones 1 and 2, and also the transformed morphology of clone 2 (panels i and l), consistent with p38 negatively regulating ENTV-1 transformation.
https://static-content.springer.com/image/art%3A10.1007%2Fs11262-007-0193-x/MediaObjects/11262_2007_193_Fig3_HTML.gif
Fig. 3

Morphological changes in transformed RK3E cells treated with MEK and p38inhibitors. RK3E cells transformed with pCMV3ΔGP(JSRV–FLAG) or pCMV3ΔGP(ENTV–FLAG) and parental RK3E cells were treated with DMSO (vehicle) ((a), (d), (g) and (j)), 5 μM PD98059 ((b), (e), (h) and (k)) or 3 μM SB203580 ((c), (f), (i) and (l)). Transformed cells were morphologically reversed ((e) and (h)) or grew slower (k) than parental RK3E cells by treatment with PD98059. Morphological changes—increasing numbers of rounded and refractile cells—were observed in only transformed cells (l) or transformed cells grew much faster than parental RK3E cells ((f) and (i)) after treatment with SB203580. Photographs were taken 24 h after treatment with inhibitors. Magnification 100x in all the panels

Role of G-proteins in ENTV-1 transformation

We previously investigated the role of the Ras family of G-proteins in JSRV-induced transformation, given the crucial role of MEK-1 in transformation [17]. We found that inhibition of H- and N-Ras with the inhibitor FTI-277 completed inhibited transformation of NIH-3T3 fibroblasts, but it partially inhibited transformation in RK3E cells [17]. As shown in Table 3, FTI-277 partially inhibited (35–40% reduction) transformation by ENTV-1 Env, similar to the inhibition of JSRV Env transformation. The fact that FTI-277 partially inhibited ENTV-1 transformation indicated that other molecules in addition to H-/N-Ras also signal through MEK-1 in transformation.
Table 3

Effects of small GTPase inhibitors on transformation of RK3E cellsa

 

Experiment 1

Experiment 2

   

Inhibition (%)

FTI-277 (μM)

0

5

0

5

       

pcDNA3.1(-)

0

0

0

0

       

pCMV3ΔGP(JSRV)

1112

724

1528

992

      

35.0 ± 0.1

pCMV3ΔGP(ENTV)

1744

996

1816

1128

      

40.4 ± 2.5

 

Experiment 1

Experiment 2

Experiment 3

Experiment 4

Experiment 5

Inhibition (%)

NSC23766 (μM)

0

10

0

10

0

10

0

10

0

10

 

pcDNA3.1(-)

0

0

0

0

0

0

0

0

0

0

 

Raf-CAAX

428

368

n.t.

n.t.

1308

1192

n.t.

n.t.

n.t.

n.t.

11.5 ± 2.6

pCMV3ΔGP(JSRV)

60

24

720

444

900

596

736

496

884

476

42.2 ± 8.6

pCMV3ΔGP(ENTV)

n.t.

n.t.

n.t.

n.t.

n.t.

n.t.

684

428

612

380

37.6 ± 0.3

aRK3E cells were transfected with the 5 μg plasmid DNAs in 6 cm dishes as described in Materials and Methods, and number of transformed foci were scored at day 17 (FTI-277) and day 19 (Exp.1), day 14 (Exp.2), day 15 (Exp.3, 4, and 5) (NSC23766) after transfection. Inhibitors were added daily at the concentrations indicated

Another G-protein that has been implicated in transformation is Rac1. Therefore, we tested the effects of the Rac1 inhibitor NSC23766 [32] on ENTV-1 and JSRV transformation as shown in Table 3. NSC23766 partially inhibited transformation by ENTV-1 and JSRV (37–42%), indicating a role for Rac1 in transformation by these two viral Env proteins. As expected, transformation by an activated Raf proto-oncogene (Raf-CAAX) was not significantly affected by NSC23766, since signaling downstream from Raf (through MEK to MAPK) would be expected to be independent of Rac1.

As an independent test for the roles of H- or N-Ras and Rac1 in ENTV-1 and JSRV transformation, co-transfections with dominant negative forms of H-ras (H-ras [17N]) and Rac1 (Rac1 [17N]) were carried out, as shown in Table 4. The dominant negative Ras or Rac1 expression plasmids were co-transfected with the JSRV or ENTV-1 Env expression plasmids at a ratio of 1:1. In general, the dominant negative H-Ras and Rac1 plasmids showed equivalent (partial) inhibition of ENTV-1 and JSRV transformation compared to the small molecule inhibitors. These results supported the conclusions that both H-Ras and Rac1 participate in JSRV transformation. Moreover, the fact that H-Ras(17N) inhibited transformation as efficiently as FTI-277 indicated that the predominant signaling is through H-Ras rather than N-Ras.
Table 4

Effect of dominant negative GTPase molecules of on transformation of RK3E cellsa

 

Experiment

Inhibition (%)

1

2

3

4

pcDNA3.1(-)

0

0

0

0

 

Rac1(17N)

0

0

0

0

 

H-ras(17N)

n.t.

0

0

0

 

pCMV3ΔGP(JSRV) + pcDNA3.1(-)

672

364

556

728

 

pCMV3ΔGP(JSRV) + Rac1(17N)

416

180

292

320

48.0 ± 5.2

pCMV3ΔGP(JSRV) + H-ras(17N)

n.t.

172

284

368

50.4 ± 1.6

pCMV3ΔGP(ENTV) + pcDNA3.1(-)

n.t.

n.t.

804

864

 

pCMV3ΔGP(ENTV) + Rac1(17N)

n.t.

n.t.

464

512

41.5 ± 0.8

pCMV3ΔGP(ENTV) + H-ras(17N)

n.t.

n.t.

508

544

37.0 ± 0.1

aRK3E cells were co-transfected with two plasmids (2.5 μg plasmid DNA each) in 6 cm dishes as described in Materials and Methods, and number of transformed foci were scored at day 15 after transfection

Discussion

In this report, signal transduction pathways involved in transformation by ENTV-1 Env protein in the RK3E rat epithelial cell line were studied. Similar approaches to those used to study signal transduction in JSRV Env transformation were employed, given the evolutionary relationship of these two viruses and the fact that they both transform secretory epithelial cells of the airways. Our previous studies on JSRV transformation had implicated the Ras–MEK–MAPK and PI3K–Akt–mTOR pathways [17], and the same pathways were implicated in ENTV-1 transformation in these studies. In particular, signaling through MEK was essential for transformation since the MEK-1/2 inhibitor PD90859 strongly inhibited transformation. Similar to the situation with JSRV, the H-/N-Ras inhibitor FTI-277 partially inhibited ENTV-1 transformation in RK3E cells, which indicated that additional pathways to MEK activation existed. In the case of JSRV, we showed that signaling through K-Ras accounted for at least part of the FTI-277 resistant transformation [17]. In RK3E cells transformed by ENTV-1 inhibition by FTI-277 and the K-ras inhibitor GGTI-298 was additive (combined inhibition 70%, not shown), indicating that signaling through K-ras or another GGTI-298 sensitive G-protein provided an alternate pathway to transformation from H-ras. ENTV-1 transformation was also strongly enhanced by the p38 MAPK inhibitor SB203580, similar to the situation for JSRV transformation of RK3E cells. For JSRV, we found that inhibition of p38 resulted in enhanced phosphorylation of ERK-1/2 MAPK, perhaps resulting from inhibition of MEK phosphatase [17]. It seems likely that the negative effect of p38 on ENTV-1 transformation might employ a similar mechanism, although the exact details remain to be elucidated. Involvement of the PI3K–Akt–mTOR pathway in ENTV-1 transformation was also evident, since rapamycin also inhibited transformation; the inhibition was somewhat stronger for ENTV-1 than for JSRV (88% vs. 75%), indicating that this pathway may be more important for transformation of these cells by ENTV-1 than for JSRV.

The involvement of these signaling pathways in ENTV-1 transformation was also indicated by studying RK3E clones transformed by an epitope-tagged ENTV-1 Env. The MEK inhibitor PD90859 partially reversed the transformed phenotype or slowed the growth rate of the cells, while the p38 inhibitor SB203580 potentiated the transformed phenotype and growth rates. In addition, we have previously reported that p44/42 MAPK activation (phosphorylation) occurs in naturally occurring ENA and OPA tumors as observed by immunohistochemistry [17], and elevated p44/42 phosphorylation was also observed in experimentally occurring OPA tumors by JSRV Env only [19]. Additional immunohistochemical studies of ENA and OPA tumors revealed that Raf–MEK–MAPK activation was detected in both cases [26].

As mentioned above, the H-/N-Ras inhibitor FTI-277 partially inhibited ENTV-1 transformation of RK3E cells, similar to its effects on JSRV transformation. In contrast, for JSRV Env, FTI-277 completed inhibited transformation of NIH-3T3 cells. Thus, signaling through to MEK and MAPK for JSRV transformation was largely downstream of H-/N-Ras in NIH-3T3 cells, but other additional molecules were involved in RK3E cells. In these experiments, co-transfection with dominant negative H-ras(17N) showed inhibition of ENTV-1 and JSRV transformation equivalent to FTI-277, indicating that the effect of FTI-277 was largely on H-ras. We also considered that other members of the Ras (small GTPase) superfamily might also be involved in ENTV-1 or JSRV transformation. These include K-, M-, N-, and R-ras, the Rho family, and Rac proteins. The Rho family consists of 22 genes encoding at least 25 proteins [33]; these proteins also have the CAAX motif at the C-terminus that would allow farnesyl or geranylgeranyl modifications at the cysteine residue. They should, therefore, be sensitive to the inhibitors FTI-277 (farnesyl transferase inhibitor) or GGTI-298 (geranylgeranyl transferase inhibitor), although as described above, the inhibition by FTI-277 appears largely attributable to H-ras.

Recently, a small molecule inhibitor of Rac1, NSC23766 [32], has become available, and we studied its effects on ENTV-1 and JSRV transformation for the first time here. NSC23766 partially inhibited transformation by both of these Env proteins, and co-transfection with a dominant negative Rac1(17N) expression plasmid showed equivalent inhibition. These experiments indicate that signaling through Rac1 is also involved in ENTV-1 and JSRV transformation, although the mechanism remains to be determined. In transformation of NIH-3T3 cells by activated H-ras, it has been reported that Rac1 can function downstream of activated H-Ras, in a signaling pathway parallel to Raf–MEK–MAPK [34]. Thus, one possible mechanism for Rac1 in ENTV-1 and JSRV transformation could be parallel signaling downstream from H-Ras (Fig. 4). Alternatively, Rac1 might signal for transformation independently of H-Ras. Recently, it has been reported that H-ras activates the MKK3/6-p38 pathway via Rac1 in MCF-10A human breast epithelial cells [35], and p38 is activated by a Rac–MEKK3–MKK3 pathway in human 293 cells transiently transfected with osmosensing scaffold for MEKK3 (OSM) [36]. This raises the possibility that the activation of p38 in JSRV-transformed cells could result from signaling from H-ras through Rac1. However, inhibition of p38 enhances transformation by ENTV-1 and JSRV, while inhibition of Rac1 inhibits it. These experiments would be facilitated if activated forms of Rac1 or Rho transformed RK3E cells. However, Rac1(115I) and RhoA(63L) [30], both of which weakly transform NIH-3T3 cells, did not transform RK3E cells.
https://static-content.springer.com/image/art%3A10.1007%2Fs11262-007-0193-x/MediaObjects/11262_2007_193_Fig4_HTML.gif
Fig. 4

Signaling pathways involved in transformation of epithelial cells with ovine β-retroviral Env proteins. The involvements of different signaling molecules in transformation by JSRV or ENTV-1 Env are shown. They are similar to our previous conclusions [27], and the possible role of Rac1 is indicated. Question marks indicate where the proteins are unknown, or where potentially more than one pathway is involved. Transformation results from integration of signals through different pathways

While the JSRV and ENTV-1 genomes share substantial sequence homology, the cytoplasmic tails of the Env TM proteins are significantly different [25]. We and others have shown that the cytoplasmic tail of JSRV TM is essential for transformation [11, 12, 249, 11, 12, 14, 16, 24]. Thus it is interesting that despite the amino acid divergence in the cytoplasmic tails of JSRV and ENTV-1 TM proteins, the patterns of signal transduction for transformation are essentially the same. It seems likely that key structural features and perhaps amino acid residues in the cytoplasmic tails are conserved, so that they contact and activate similar signaling molecules, resulting in activation of the same signaling pathways. For instance, we and others have highlighted the importance of a single tyrosine residue in the cytoplasmic tail of JSRV TM, Y590 [6, 7, 9, 11, 12, 14, 1618, 24]. The Y590 is conserved in the ENTV-1 cytoplasmic tail, and mutation of this residue greatly reduces transformation [6, 9, 16]; in contrast, mutation of two other tyrosine residues in the ENTV-1 TM (Y592 and Y596) had relatively less effect [6]. We have conducted alanine scanning mutagenesis of the JSRV cytoplasmic tail [24], as well as preliminary NMR structural studies (M. Cocco, N. Maeda and H. Fan, unpublished). The results from these structure-function studies, combined with comparisons with the ENTV-1 cytoplasmic tail might provide an additional insight into the mechanism of transformation. Ultimately, identification of the cellular proteins that interact with the cytoplasmic tail of the JSRV and ENTV-1 TM proteins will be key to linking these structural studies with the signal transduction pathways involved in transformation.

Finally, the results in this study are interesting with regard to the relative disease specificities of ENTV-1 (causing ENA of the ethmoid turbinate cells) and JSRV (causing OPA of type II pneumocytes and Clara cells). The fact that the signal transduction pathways activated during transformation by these two viral Env proteins are the same support the notion that the disease specificity does not result from their specificities for transforming different cell types. This is consistent with the fact that inoculation of an AAV vector expressing the ENTV-1 Env protein in mice results in lung tumors with equivalent histopathology to those induced by an AAV vector expressing the JSRV Env protein. At the same time, we have reported that the JSRV LTR functions efficiently in murine lung epithelial cell lines in transient transfection assays [37]. Additional studies identified binding motifs in the JSRV LTR for transcription factors known to be important for expression of cellular genes in lung epithelial cells, most notably HNF-3, NF-1 and C/EBP [38, 39]. Binding sites for these factors are largely absent from the ENTV-1 LTR [40], supporting the notion that the ENTV-1 LTR would not be highly active in lung epithelial cells. Thus the tissue specificity of JSRV vs. ENTV-1-induced disease may be strongly influenced by the transcriptional specificities of the respective viral LTRs, and not by the signaling pathways activated by the respective Env proteins.

Acknowledgments

This work was supported by grant R01CA94188 from the National Institutes of Health (to H.F.). Support of the DNA sequencing shared resource of the UCI Chao Family Comprehensive Cancer Center is acknowledged.

Copyright information

© Springer Science+Business Media, LLC 2008