Effects of EGF-R on Transcription Factor Activation in Hematopoietic Cells: EGF-R Activates Multiple Transcription Factors Which Induce Proliferation in the Absence of Autocrine Cytokines.

Abstract

v-ErbB is an oncogene related to the Epidermal Growth Factor Receptor (EGF-R) that was initially discovered in the genome of avian erythoblastosis virus. v-ErbB will abrogate the requirement of erythroid progenitor cells for erythropoietin and stem cell factor and block terminal differentiation. EGF-R overexpression has been observed in many pathological situations and the EGF-R gene is amplified in certain tumors. Moreover, there is a truncated form of EGF-R referred to as EGFvIII which resembles v-ErbB in biological properties. One problem frequently encountered in studying the effects of EGF-R overexpression in many tumors is that EGF-R expression is often constitutive and in the presence of increased expression of other oncogenes or in the absence of certain tumor suppressor genes. To circumvent these problems, we subcloned v-ErbB into a vector which contained the estrogen receptor hormone binding domain which renders the v-ErbB protein dependent upon the addition of beta-estradiol or 4 OH tamoxifen for activity. The v-ErbB:ER oncogene will conditionally abrogate the cytokine dependence of human (TF-1) and murine (FL5.12 and FDC-P1) hematopoietic cells efficiently. This construct has allowed us to examine the transcription factors pathways activated by v-ErbB:ER in hematopoietic cells which are required for proliferation in the absence of previously required cytokines. By determining which signal transduction pathways were activated in response to either v-ErbB:ER activation or IL-3 addition in the presence and absence of specific small molecular weight membrane permeable kinase inhibitors, we could ascertain that v-ErbB:ER expression activated the STAT5, Elk, CREB, Jun, and Forkhead (Foxo) family of transcription factors in FL/v-ErbB:ER cells. The activation of these transcription factors was blocked by the respective kinase inhibitors. Thus v-ErbB:ER activated a broad spectrum of transcription factors. Treatment of v-ErbB:ER cells with the EGF-R inhibitor AG1478 very efficiently induced apoptosis in these cells at 100 to 1000 fold lower concentrations than MEK, PI3K or JNK inhibitors and activation of all these transcription factor inhibitors was inhibited. In contrast, when the cells were treated with MEK, PI3K or JNK inhibitors, only the transcription factors specific for the individual pathways were inhibited. FL5.12 cells conditionally transformed to grow in response to activated Raf and Akt, (FL/Akt:ER+Raf-1:AR) were also isolated. Activation of STAT5 by either Raf or Akt did not occur in FL/Akt:ER+Raf-1:AR cells, but did occur when the cells were treated with IL-3. Furthermore, Elk activation occurred in response to Raf activation but not IL-3 stimulation in the FL/Akt:ER+Raf-1:AR cells which grew in response to Raf and Akt. Thus oncogenes such as v-ErbB:ER, which are more effective in their ability to transform hematopoietic cells than oncogenes such as Raf and Akt, can induce multiple transcription factors, only some of which are required for growth in tissue culture systems.