Cell Type-specific Modulation of Cell Growth by Transforming Growth Factor β1 Does Not Correlate with Mitogen-activated Protein Kinase Activation

Abstract

Transforming growth factor ␤1 (TGF-␤1) is a multifunctional cytokine that positively or negatively regulates the proliferation of various types of cells. In this study we have examined whether or not the activation of the mitogen-activated protein (MAP) kinases is involved in the transduction of cell growth modulation signals of TGF-␤1, as MAP kinase activity is known to be closely associated with cell cycle progression. Although TGF-␤1 stimulated the growth of quiescent Balb 3T3 and Swiss 3T3 cells, it failed to detectably stimulate the tyrosine phosphorylation and activation of the 41-and 43-kDa MAP kinases at any time point up to the reinitiation of DNA replication. TGF-␤1 also failed to stimulate the expression of the c-fos gene. Furthermore, TGF-␤1 synergistically enhanced the mitogenic action of epidermal growth factor (EGF) without affecting EGF-induced MAP kinase activation in these fibroblasts, and it inhibited the EGF-stimulated proliferation of mouse keratinocytes (PAM212) without inhibiting EGF-induced MAP kinase activation. Thus, the ability of TGF-␤1 to modulate cell proliferation is apparently not associated with the activation of MAP kinases. In this respect, TGF-␤1 is clearly distinct from the majority, if not all, of peptide growth factors, such as platelet-derived growth factor and EGF, whose ability to modulate cell proliferation is closely associated with the activation of MAP kinases. These results also suggest that the activation of MAP kinases is not an absolute requirement for growth factor-stimulated mitogenesis.

TGF-β SIGNAL TRANSDUCTION

“…Several groups investigating whether TGF-β action affects the Erk subfamily of MAP kinases have reported activation (245), inhibition (246,247), or no change (248) in the activity of these kinases after TGF-β treatment. A novel member of this family, TAK1 (TGF-β-activated kinase 1) was cloned based on its ability to activate a MAP kinase cascade in yeast (249).…”

Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors

“…A polyclonal anti-MAP kinase antibody was raised against residues 299 ± 321 (RIEVEQALAHPYLEQYYDPSDEP) of 41-kDa MAP kinase (ERK2) and was shown to recognize both the 41-and 43-kDa MAP kinases (Chatani et al, 1992;Chatani et al, 1995). The polyclonal anti-Raf-1 antibody (C-12) and polyclonal anti-p90 rsk antibody (C-21) were obtained from Santa Cruz Biotechnology, Inc.…”

Activation of the 41/43 kDa mitogen-activated protein kinase signaling pathway is required for hepatocyte growth factor-induced cell scattering

“…EGF puri®ed from mouse submaxillary glands was purchased from Toyobo Co (Osaka, Japan), and PMA was from Sigma Chemical Co. A polyclonal anti-MAP kinase antibody was raised against residues 299 ± 321 (RIEVEQALAHPYLEQYYDPSDEP) of 41 kDa MAP kinase (ERK2) and was shown to recognize both the 43 kDa and 41 kDa MAP kinases (Chatani et al, 1992(Chatani et al, , 1995. The polyclonal anti-Raf-1 antibody (C-12) was obtained from Santa Cruz Biotechnology, Inc, the monoclonal anti-MEK1 antibody was from Transduction Laboratories, and the polyclonal anti-p90 rsk antibody (rsk-CT), p90 rsk substrate peptide (S6 peptide; RRRLSSLRA), monoclonal anti-phosphotyrosine antibody (4G10), polyclonal anti-PI3-kinase antibody and GST-MEK1 K97A were from Upstate Biotechnology, Inc.…”

“…Cell lysates (10 ± 100 mg of protein) were then immunoprecipitated by incubating for 3 h at 48C with each of respective antibodies preadsorbed to protein-A Sepharose (Pharmacia Biotech Inc). For the immunoprecipitation of 41/ 43 kDa MAP kinases, cell lysates were pretreated with 0.1% SDS for 20 min at 48C, diluted 1 : 10 in cell lysis buer, and then the antibody was added (Chatani et al, 1992(Chatani et al, , 1995. The immunoprecipitates were washed three times with each of the respective cell lysis buers.…”

“…A requirement for the activation of the 41/43 kDa MAP kinase signaling pathway for cell proliferation induced by the majority of, but not all (Chatani et al, 1995) mitogens has already been established (Nishida and Gotoh, 1993;Cobb and Goldsmith, 1995;Seger and Krebs, 1995). In this respect, EGF treatment of MDCK cells also induced the activation of MEK, 41/ 43 kDa MAP kinases and p90 rsk with similar timecourse pro®les, but to lesser extents (60 ± 70%), as those observed in HGF-stimulated MDCK cells.…”