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. 1999 May 11;96(10):5716-21.
doi: 10.1073/pnas.96.10.5716.

Heparin-binding epidermal growth factor-like growth factor, a v-Jun target gene, induces oncogenic transformation

S l Fu  1 , I Bottoli, M Goller, P K Vogt
Affiliations

Heparin-binding epidermal growth factor-like growth factor, a v-Jun target gene, induces oncogenic transformation

S l Fu et al. Proc Natl Acad Sci U S A. .

Abstract

Jun is a transcription factor belonging to the activator protein 1 family. A mutated version of Jun (v-Jun) transduced by the avian retrovirus ASV17 induces oncogenic transformation in avian cell cultures and sarcomas in young galliform birds. The oncogenicity of Jun probably results from transcriptional deregulation of v-Jun-responsive target genes. Here we describe the identification and characterization of a growth-related v-Jun target, a homolog of heparin-binding epidermal growth factor-like growth factor (HB-EGF). HB-EGF is strongly expressed in chicken embryo fibroblasts (CEF) transformed by v-Jun. HB-EGF expression is not detectable or is marginal in nontransformed CEF. Using a hormone-inducible Jun-estrogen receptor chimera, we found that HB-EGF expression is correlated with v-Jun activity. In this system, induction of v-Jun is followed within 1 hr by elevated levels of HB-EGF. In CEF infected with various Jun mutants, HB-EGF expression is correlated with the oncogenic potency of the mutant. Constitutive expression of HB-EGF conveys to CEF the ability to grow in soft agar and to form multilayered foci of transformed cells on a solid substrate. These observations suggest that HB-EGF is an effector of Jun-induced oncogenic transformation.

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Figures

Figure 1
Figure 1
Expression of VJT-6 is regulated by v-Jun. (A) v-Jun-transformed CEF overexpress VJT-6. Two micrograms of poly(A)+ RNA from CEF infected with the RCAS(A) vector or RCAS(A)v-Jun was analyzed by Northern blot and autoradiographed. (B) Induction of VJT-6 by the estrogen-regulated Jun-estrogen receptor chimera ΔVJ-hER. Twenty micrograms of total RNA from CEF expressing the hormone-binding domain of the human estrogen receptor (hER), v-Jun (VJ1), or ΔVJ-hER was used for Northern blots. −, Control treatment (10 μl of EtOH) for 48 hr; +, exposure to 2 μM estrogen in EtOH for 48 hr; and +/−, 48 hr of estrogen treatment, followed by 48 hr without estrogen. (C) Time course of VJT-6 induction by ΔVJ-hER. CEF infected with ΔVJ-hER were treated with estrogen for various time periods. Twenty micrograms of total RNA from each indicated time point was analyzed by Northern blot. For A–C, blots were probed with 32P-labeled VJT-6 cDNA. Molecular markers are on the left (×1,000). Control blots for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) show equal loading of the lanes. B and C were generated with different exposure times; quantitative aspects of VJT-6 induction are not comparable between B and C.
Figure 2
Figure 2
Alignment of putative chicken HB-EGF to mammalian HB-EGF proteins and the domains of HB-EGF. The amino acid sequences of mammalian HB-EGF proteins were downloaded from the Swiss-Prot database. The numbers indicate the amino acid numbers of the putative chicken HB-EGF. The dark gray areas indicate identical amino acids, and light gray areas indicate similar amino acids. The alignment was generated with the program clustalw alignment. The arrows mark the amino acid sequence of predicted, mature-secreted HB-EGF. The black dot designates the potential glycosylation site, threonine-89. The dashed lines mark the heparin-binding sites. The transmembrane domain is underlined. Swiss-Prot database accession numbers are Q99075 (human HB-EGF), Q061767 (rat HB-EGF), and Q06186 (mouse HB-EGF). The chicken HB-EGF has been deposited in GenBank (accession no. AF131224).
Figure 3
Figure 3
HB-EGF expression correlates with oncogenic transformation. Northern blot demonstrating the expression of HB-EGF from cells transfected with indicated Jun mutants. Twenty micrograms of total RNA from CEF infected by different Jun mutants was analyzed with HB-EGF or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as probes. The transformation potential of these mutants is divided into three categories: ++, strongly transforming constructs, focus-forming activity equivalent to or higher than v-Jun and morphological transformation comparable to that induced by v-Jun; +, weakly transforming constructs, forming 5–15% the number of foci of v-Jun per μg DNA and inducing less pronounced morphological changes; −, nontransforming constructs.
Figure 4
Figure 4
Transformed cell foci induced by HB-EGF. CEF were transfected with 0.5 μg of DNA per well, overlaid with nutrient agar, and stained 12 days posttransfection.
Figure 5
Figure 5
Anchorage-independent growth of HB-EGF-expressing CEF. CEF expressing vector RCAS(A), positive control v-Jun, or HB-EGF were seeded in soft-agar assays as described in Materials and Methods. Experiments were repeated four times, and one typical experiment is shown.
Figure 6
Figure 6
Morphology of HB-EGF-transformed cells. CEF expressing RCAS(A), RCAS(A)v-Jun, or RCAS(A)HB-EGF were grown into mass culture and photographed 3 weeks postinfection with phase-contrast optics at ×16 objective lens magnification.
Figure 7
Figure 7
HB-EGF expression in CEF transformed by various oncogenes. CEF were infected with various oncogenes. Twenty micrograms of total RNA was analyzed in Northern blots by using HB-EGF or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as probes.
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