Insulin-like growth factor I synergizes with interleukin 4 for hematopoietic cell proliferation independent of insulin receptor substrate expression.

In the present study, we investigated the potential role of insulin-like growth factor I (IGF-I) receptor (IGF-IR) in cell proliferation by overexpressing it in 32D myeloid progenitor cells. The overexpression of IGF-IR caused the transfectants to proliferate in response to IGF-I in the absence of insulin receptor substrate (IRS) expression. The activation of overexpressed wild-type IGF-IR, but not that of an ATP-binding mutant of IGF-IR, resulted in the increased tyrosine phosphorylation of several intracellular proteins, including SHC, Src homology 2-containing inositol-5-phosphatase, protein kinase C-delta, and Erk2. Grb2 association with SHC and mitogen-activated protein kinase (MAPK) activity was also enhanced in response to IGF-I stimulation. Interestingly, the stimulation of the IGF-IR transfectants with interleukin 4 (IL-4) also resulted in strong mitogenesis independent of IRS expression. Moreover, IGF-I and/or IL-4 induced long-term cell growth of the IGF-IR transfectants. IL-4 was able to synergize with IGF-I for DNA synthesis, even in the parental 32D cells and a pro-B-cell line, Baf3, indicating the physiological importance of the two growth factors in hematopoietic cell proliferation. IL-4 stimulation of the IGF-IR transfectants resulted in enhanced tyrosine phosphorylation of SHC, Erk2, and signal transducer and activator of transcription 6 (STAT6) proteins. Both IL-4 and IGF-I were able to induce c-myc early response gene expression, and this expression was maximal in the presence of both factors. Finally, we demonstrated that a MAPK kinase inhibitor was able to suppress mitogenesis of the IGF-IR transfectants in response to IGF-I and/or IL-4. Together, our results suggest that IL-4 synergizes with IGF-I for hematopoietic cell proliferation, likely through cross talk between SHC/Grb2/MAPK and STAT6 pathways and through c-myc gene up-regulation.

Protein kinase C-delta is an important signaling molecule in insulin-like growth factor I receptor-mediated cell transformation.

To investigate the potential role of protein kinase C-delta (PKC-delta) in insulin-like growth factor I receptor (IGF-IR)-mediated cell transformation, an oncogenic gag-IGF-IR beta-fusion receptor lacking the entire extracellular domain, which was designated NM1, and a full-length IGF-IR were coexpressed with either wild-type PKC-delta (PKC-deltaWT) or an ATP-binding mutant of PKC-delta (PKC-deltaK376R) in NIH 3T3 fibroblasts. While overexpression of PKC-deltaWT did not affect NM1- and IGF-IR-induced focus and colony formation of NIH 3T3 cells, expression of PKC-deltaK376R severely impaired these events. In contrast, NM1-mediated cell growth in monolayer was not affected by coexpressing PKC-deltaK376R. PKC-deltaWT and PKC-deltaK376R were constitutively phosphorylated on a tyrosine residue(s) in the NM1- and IGF-IR-expressing cells and were associated with them in an IGF-I-independent manner. Activated IGF-IR was able to phosphorylate purified PKC-delta in vitro and stimulated its kinase activity. Furthermore, the level of endogenous PKC-delta protein was up-regulated through transcriptional activation in response to long-term IGF-IR activation. Taken together, our results demonstrate that PKC-delta plays an important role in IGF-IR-mediated cell transformation, probably via association of the receptor with PKC-delta and its activation through protein up-regulation and tyrosine phosphorylation. Competition with endogenous PKC-delta for NM1 and IGF-IR association by PKC-deltaK376R is probably an important mechanism underlying the PKC-deltaK376R-mediated inhibition of cell transformation by NM1 and IGF-IR.

Activation of insulin-like growth factor I receptor signaling pathway is critical for mouse plasma cell tumor growth.

Plasma cell neoplasia in humans generally occurs as multiple myeloma, an incurable form of cancer. Tumors with marked similarity can be induced in mice by a variety of agents, including chemicals, silicone, and oncogene-containing retroviruses, suggesting the use of murine tumors as an informative model to study plasma cell disease. Herein, we have focused on the role of insulin-like growth factor I receptor (IGF-IR) signaling in the development of plasma cell disease. The insulin receptor substrate 2/phosphatidylinositol 3'-kinase/p70S6K pathway was found to be either constitutively or IGF-I-dependently activated in all plasma cell tumors. Biological relevance was demonstrated in that plasma cell lines with up-regulated IGF-IR expression levels exhibited mitogenic responses to IGF-I. More importantly, expression of a dominant-negative mutant of IGF-IR in these lines strongly suppressed tumorigenesis in vivo. Taken together, these results demonstrate that up-regulation and activation of IGF-IR and the downstream signaling pathway involving insulin receptor substrate 2, phosphatidylinositol 3'-kinase, and p70S6K may play an important role in the development of a broad spectrum of plasma cell tumors.

Protein kinase C-alpha overexpression stimulates Akt activity and suppresses apoptosis induced by interleukin 3 withdrawal.

To investigate the role of protein kinase C (PKC) in apoptotic signaling induced by cytokine withdrawal, we expressed PKC-alpha, -delta and -epsilon individually in the 32D myeloid progenitor cells. The parental and PKC-delta- and PKC-epsilon-transfected 32D cells underwent apoptosis within 24 h in the absence of interleukin 3. In contrast, expression of PKC-alpha inhibited the onset of apoptosis as determined by genomic DNA fragmentation and flow cytometric analysis. Correlating with the inhibition of apoptosis, PKC-alpha transfectants exhibited increased activity of the endogenous Akt serine/threonine kinase. Furthermore, PKC-alpha, but not PKC-delta or -epsilon, specifically activated overexpressed Akt. PKC-alpha-induced Akt activity was partially dependent on phosphoinositol 3' kinase (PI 3'K) since a PI 3'K inhibitor was able to suppress PKC-alpha-induced Akt activation. Both basal and interleukin 3-stimulated phosphorylation of Akt on serine 473 was enhanced in the PKC-alpha and Akt contransfectants. Coexpression of wild type Akt and PKC-alpha resulted in greater suppression of apoptosis than PKC-alpha expression alone. Together, our results demonstrate that suppression of apoptosis by PKC-alpha correlates with its ability of activating endogenous Akt. Furthermore, activation of overexpressed Akt by PKC-alpha is consistent with their synergistic effect on suppressing apoptosis, providing the strong evidence of cross talk between Akt and PKC-alpha.