Hyaluronidase 2 negatively regulates RON receptor tyrosine kinase and mediates transformation of epithelial cells by jaagsiekte sheep retrovirus

Abstract

The candidate tumor-suppressor gene hyaluronidase 2 (HYAL2) encodes a glycosylphosphatidylinositol-anchored cell-surface protein that serves as an entry receptor for jaagsiekte sheep retrovirus, a virus that causes contagious lung cancer in sheep that is morphologically similar to human bronchioloalveolar carcinoma. The viral envelope (Env) protein alone can transform cultured cells, and we hypothesized that Env could bind and sequester the HYAL2 receptor and thus liberate a potential oncogenic factor bound and negatively controlled by HYAL2. Here we show that the HYAL2 receptor protein is associated with the RON receptor tyrosine kinase (also called MST1R or Stk in the mouse), rendering it functionally silent. In human cells expressing a jaagsiekte sheep retrovirus Env transgene, the Env protein physically associates with HYAL2. RON liberated from the association with HYAL2 becomes functionally active and consequently activates the Akt and mitogen-activated protein kinase pathways leading to oncogenic transformation of immortalized human bronchial epithelial cells. We find activated RON in a subset of human bronchioloalveolar carcinoma tumors, suggesting RON involvement in this type of human lung cancer.

Figure 1

Effects of JSRV Env expression in BEAS-2B cells. BEAS-2B cells were transfected with pHA-Jenv (Env) or the empty pCR3.1 expression vector (Control) and grown in the presence of G418 (see Materials and Methods). (A) Cells were photographed 6 weeks after transfection. The arrow points to a transformed focus. (B) RT-PCR was used to detect the presence of Env mRNA in transfected cells (Left), and immunofluorescence staining was used to detect the expression and subcellular localization of JSRV Env protein (Center and Right). For staining, cells grown on coverslips were fixed, permeabilized, and processed as described (18). Cells were stained with anti-hemagglutinin antibody then secondary goat anti-rabbit antibody conjugated with rhodamine and were counterstained with 4′,6-diamidino-2-phenylindole. (C) RON receptor was immunoprecipitated (IP) from cell lysates by anti-RON antibodies. RON tyrosine phosphorylation was detected by Western blotting (WB) with anti-PY antibodies (Top). RON was detected by using anti-RON antibodies (Middle). The two bands represent mature RON (lower band) and its intracellular precursor (upper band). HYAL2 was detected with anti-HYAL2 antibodies (Bottom). IP control, immunoprecipitations of BEAS-2B control cell lysates with nonspecific rabbit IgG antibodies in place of RON antibodies followed by Western blotting as indicated. (D) Activation of Akt and MAPK in cell lysates was determined by Western blotting with anti-phospho-Akt and anti-phospho-MAPK antibodies. The amount of Akt and MAPK (the two bands represent p42 and p44 ERK1/ERK2) was determined by reprobing of the membrane with anti-Akt and anti-MAPK antibodies. The positions of molecular mass markers are indicated at right.

Figure 2

Reversion of Env-induced transformation of BEAS-2B cells by stable expression of RONkd. BEAS-2B cells were transfected with equal amounts of the following plasmids: Control, pCR3.1 and pMSCV-puro-EGFP; Env, pHA-Jenv and pMSCV-puro-EGFP; Env/RONkd, pHA-Jenv and pMSCV-puro-RONkd. Transfected cells were cultured in the presence of G418 and puromycin to select for the expression of both plasmids. (A) Cells photographed 6 weeks after transfection. The arrow indicates a focus of Env-transformed cells. (B) Control, Env, and Env/RONkd cells were fixed and stained with Giemsa dye, and foci were counted by using a microscope. Bars represent the number (mean ± SE) of foci counted in 25 fields (×16 magnification) in three independent experiments. (C) Cells were seeded at 2 × 10⁴ cells per well in duplicate wells of 24-well plates. Cell numbers were measured at the indicated time points by using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cell-growth determination kit (Sigma). The number of cells was determined from an MTT calibration curve generated for each cell line. Each experimental point represents the mean ± SE of three independent experiments. (D) RON, Akt, MAPK, and their tyrosine-phosphorylated forms were detected as described for Fig. 1. The intensity of the RON bands is noticeably less in the Env-transformed cells as compared with Fig. 1C because of rapid turnover and recycling of activated RON during continuous culture of transformed cells. The dense RON band in Env/RONkd cells represents overexpressed RONkd. The positions of molecular mass markers are indicated at right. IP, immunoprecipitated; WB, Western blot.

Figure 3

Effects of Env and HYAL2 expression on RON activation in RE7 epithelial cells. RE7 cells were transiently transfected with an empty vector (Control), pJenv-V5 (Env), pFlag-HYAL2 (HYAL2), or both pJenv-V5 and pFlag-HYAL2 (Env+HYAL2). Two days later cells were incubated overnight in DMEM without serum in the presence of the proteasomal inhibitor ALLN (see Materials and Methods). After incubation the cells were lysed and analyzed by immunoprecipitation (IP) and Western blotting (WB). (A and B) RON, MAPK, and their tyrosine-phosphorylated forms were detected as described for Fig. 1. (C) Env was immunoprecipitated from transiently transfected RE7 cells with anti-V5 antibody. Expression was detected by Western blotting with anti-V5 antibody (Upper). The upper arrow on the left shows the position of the full-length Env protein. The lower arrow on the left points on the transmembrane domain (TM) of the Env protein generated by cleavage of the Env protein with endoplasmic proteases. Tyrosine phosphorylation of the Env protein was probed with anti-PY antibody (Lower). The arrows on the right show the positions of Ig heavy chains (IgH). (D) HYAL2 was detected in Env precipitates with anti-HYAL2 antibodies and in RON immunoprecipitates with anti-Flag antibody. Arrows on the right show the positions of HYAL2. (E) Expression of Env was detected with anti-V5 antibody and HYAL2 was detected with anti-Flag antibody in RE7 total-cell lysates. The positions of molecular mass markers are indicated at the right in all panels.

Figure 4

Effects of Env expression in HEK293 cells. (A) HEK293 cells were transiently transfected with an empty vector (Control) or pJenv-V5 (Env) plasmid. Env protein was detected as described for Fig. 3. The upper arrow on the left indicates the position of the full-length Env protein, and the lower arrow indicates the position of the transmembrane domain of the Env protein. Tyrosine phosphorylation of the Env protein was probed with anti-PY antibody (Right). The positions of Ig heavy chain (IgH) proteins are shown. IP, immunoprecipitated; WB, Western blot. (B) HEK293 cells were transiently transfected with an empty vector (control), HYAL2, or HYAL2 and Env expression vectors. Forty-eight hours after transfection the cells were treated with or without ALLN proteasome inhibitor overnight, and total-cell lysates were prepared and analyzed as indicated. (C) HEK293 cells were transiently transfected with 5 μg of pCIneo-RON wild type, 5 μg of pFlag-HYAL2, and increasing amounts of pJenv-V5 plasmid. Two days later cells were incubated overnight in DMEM without serum in the presence of the proteasomal inhibitor ALLN (see Materials and Methods). After incubation the cells were lysed and analyzed. RON and tyrosine-phosphorylated RON was detected as described for Fig. 1. Env protein was detected in total-cell lysates with anti-V5 antibodies. Only the Env transmembrane bands are shown, but the full-length Env showed parallel changes in abundance. (D) HEK293 cells were transiently transfected as described for B. The presence of HYAL2 in RON immunoprecipitates was detected by anti-Flag antibody (Top). The presence of HYAL2 in Env immunoprecipitates was detected by anti-HYAL2 antibodies (Middle). HYAL2 was detected in total-cell lysates with anti-Flag antibody (Bottom). The positions of molecular mass markers are indicated at the right in all panels.

Figure 5

RON tyrosine phosphorylation in human BAC cell lines. RON and tyrosine-phosphorylated RON were detected as described for Fig. 1. Two milligrams of total protein was analyzed in each lane. The positions of molecular mass markers are indicated at the right. IP, immunoprecipitated; WB, Western blot.

Figure 6

Model of Env-mediated transformation of human bronchial epithelial cells. (A) BEAS-2B cells express RON receptor tyrosine kinase (Fig. 1). RON is expressed on the cell surface as an inactive dimer (25). Association of HYAL2 with RON prevents RON activation. (B) JSRV interacts with HYAL2 via the Env protein. This interaction leads to viral entry and subsequent sequestration of HYAL2 by expressed Env and intracellular degradation of HYAL2-Env complex by a proteasomal-dependent mechanism (Figs. 3D and 4 C and D). Removal of HYAL2 from the RON complex may cause conformational changes in the RON kinase domain triggering RON catalytic activity. RON constitutive activation induces activation of oncogenic pathways involving MAPK and Akt causing cell transformation. Human and animal BAC tumors are commonly composed of three types of cells, namely Clara, mucin-producing, and alveolar type II cells, none of which express RON in normal lung (26). However a common progenitor cell for these cell types, which probably gives rise to BAC, may reside in the bronchiolar compartment and may express RON (26). The heterogeneity of the differentiation phenotype within BAC lesions has been well established.