Faculty Bibliography

We investigated the role of 14-3-3sigma protein in insulin-like growth factor-I (IGF-I) receptor signaling. It has been previously shown that 14-3-3sigma negatively regulates cell cycle especially in response to p53-sensitive DNA damage. In this study we demonstrated that 14-3-3sigma is a positive mediator of IGF-I receptor-induced cell proliferation. Treatment with IGF-I increased 14-3-3sigma mRNA and protein levels about 4-fold, in a time-dependent manner in MCF-7 breast cancer cells. Preincubation with the phosphoinositide 3'-kinase inhibitor LY294002 significantly reduced the effects of IGF-I on 14-3-3sigma gene expression in these cells, suggesting that this effect of IGF-I occurs via the phosphoinositide 3'-kinase pathway. 14-3-3sigma is induced by IGF-I in MCF-7 cells, which express wild-type p53, as well as in MCF-7 cells transfected with a small interference RNA targeting duplex that reduced p53 expression levels. These results suggest that IGF-I induces 14-3-3sigma expression in a manner that is independent of p53. Using the small interference RNA strategy, we demonstrated that a 70-75% reduction of 14-3-3sigma mRNA levels resulted in a similar decrease in the effects of IGF-I on cell cycle progression and proliferation in MCF-7 cells. This effect was also associated with a reduction in IGF-I-induced cyclin D1 expression. Taken together, these results suggest that 14-3-3sigma positively mediates IGF-I-induced cell cycle progression.

Insulin-like growth factor-I (IGF-I) receptors and insulin receptors belong to the same subfamily of receptor tyrosine kinases and share a similar set of intracellular signaling pathways, despite their distinct biological actions. In the present study, we evaluated T cell death-associated gene 51 (TDAG51), which we previously identified by cDNA microarray analysis as a gene specifically induced by IGF-I. We characterized the signaling pathways by which IGF-I induces TDAG51 gene expression and the functional role of TDAG51 in IGF-I signaling in NIH-3T3 (NWTb3) cells, which overexpress the human IGF-I receptor. Treatment with IGF-I increased TDAG51 mRNA and protein levels in NWTb3 cells. This effect of IGF-I was specifically mediated by the IGF-IR, because IGF-I did not induce TDAG51 expression in NIH-3T3 cells overexpressing a dominant-negative IGF-I receptor. Through the use of specific inhibitors of various protein kinases, we found that IGF-I induced TDAG51 expression via the p38 MAPK pathway. The ERK, JNK, and phosphatidylinositol 3-kinase pathways were not involved in IGF-I-induced regulation of TDAG51. To assess the role of TDAG51 in IGF-I signaling, we used small interfering RNA (siRNA) expression vectors directed at two different target sites to reduce the level of TDAG51 protein. In cells expressing these siRNA vectors, TDAG51 protein levels were decreased by 75-80%. Furthermore, TDAG51 siRNA expression abolished the ability of IGF-I to rescue cells from serum starvation-induced apoptosis. These findings suggest that TDAG51 plays an important role in the anti-apoptotic effects of IGF-I.

One of the hallmarks of leukemic cells is their ability to proliferate and survive in the absence of exogenous growth factors (GFs). However, the molecular mechanisms used by myeloid tumor cells to escape apoptosis are not fully understood. Here we report that Myc/Raf-transformed macrophages require the transcription factor C/EBP beta to prevent cell death. In contrast to wild-type cells, C/EBP beta(-/-) macrophages were completely dependent on macrophage colony-stimulating factor or granulocyte-macrophage colony-stimulating factor for survival and displayed impaired tumorigenicity in vivo. Microarray analysis revealed that C/EBP beta-deficient cells expressed significantly reduced levels of the prosurvival factor insulin-like growth factor I (IGF-I). Overexpression of C/EBP beta stimulated transcription from the IGF-I promoter, indicating that IGF-I is a direct transcriptional target of C/EBP beta. Serological neutralization of IGF-I in C/EBP beta(+/+) tumor cell cultures induced apoptosis, showing that IGF-I functions as an autocrine survival factor in these cells. Macrophage tumor cells derived from IGF-I(-/-) mice were GF dependent, similar to C/EBP beta-deficient cells. Forced expression of either C/EBP beta or IGF-I in C/EBP beta(-/-) bone marrow cells restored Myc/Raf-induced transformation and permitted neoplastic growth without exogenous GFs. Thus, our findings demonstrate that C/EBP beta is essential for oncogenic transformation of macrophages and functions at least in part by regulating expression of the survival factor IGF-I.

PTEN is a tumor suppressor gene that is frequently mutated in human tumors. It functions primarily as a lipid phosphatase and plays a key role in the regulation of phosphatidylinositol-3'-kinase. PTEN appears to play a crucial role in modulating apoptosis by reducing the levels of PtdIns(3,4,5)P3, a phospholipid that activates AKT, a central regulator of apoptosis. To understand the role of PTEN in regulating cell proliferation and apoptosis, we stably overexpressed PTEN in PC3 cells, which are prostate cancer cells that lack PTEN. Overexpression of PTEN in two different clones inhibited cell proliferation and increased serum starvation-induced apoptosis, as compared to control cells. Interestingly, PTEN overexpression resulted in a 44-60% reduction in total insulin-like growth factor-I receptor (IGF-IR) protein levels and a 49-64% reduction in cell surface IGF-IR expression. [35S]methionine pulse experiments in PC3 cells overexpressing PTEN demonstrated that these cells synthesize significantly lower levels of the IGF-IR precursor, whereas PTEN overexpression had no effect on IGF-IR degradation. Taken together, our results show that PTEN can regulate cell proliferation and apoptosis through inhibition of IGF-IR synthesis. These results have important implications for understanding the roles of PTEN and the IGF-IR in prostate cancer cell tumorigenesis.

To determine whether circulating insulin-like growth factor (IGF)-I has a role in hematopoiesis, we examined hematologic parameters in mice with markedly reduced serum levels resulting from a liver-specific inactivation of the IGF-I gene. These mice have normal postnatal growth and development, suggesting that local production of IGF-I can maintain anabolic effects. Liver-specific IGF-I-deficient (LID) mice were compared with control littermates with regard to hematopoietic parameters. Spleen cellularity was decreased in the LID mice compared with control mice. Spleen myeloid progenitors, as determined by colony-forming units-granulocyte/monocyte (CFU-GM) and colony-forming units-high proliferative potential (CFU-HPP), were significantly decreased in the LID mice. Immune parameters, as indicated by the absolute number of B and T cells, did not significantly differ between the knockout and control mice. In contrast to the decreased cellularity and myelopoiesis in the spleen, bone marrow cellularity was not different between the 2 groups, but the total femoral content of CFU-GM and CFU-HPP was significantly increased in the LID mice. The decrease in splenic myelopoiesis was not due to the inability of progenitors to exit the bone marrow, because CFU-GM and burst-forming units-erythroid were significantly increased in the blood of LID mice compared with normal littermates. Administration of exogenous IGF-I to the LID mice for 4 days partially restored myelopoietic parameters in the spleen. Liver production of IGF-I and, therefore, normal serum levels of this hormone, although not necessary for general organ growth and development, seems necessary for survival or transition of myeloid progenitors into the spleen.

Liver IGF-1-deficient (LID) mice have a 75% reduction in circulating IGF-1 levels and, as a result, a fourfold increase in growth hormone (GH) secretion. To block GH action, LID mice were crossed with GH antagonist (GHa) transgenic mice. Inactivation of GH action in the resulting LID + GHa mice led to decreased blood glucose and insulin levels and improved peripheral insulin sensitivity. Hyperinsulinemic-euglycemic clamp studies showed that LID mice exhibit severe insulin resistance. In contrast, expression of the GH antagonist transgene in LID + GHa mice led to enhanced insulin sensitivity and increased insulin-stimulated glucose uptake in muscle and white adipose tissue. Interestingly, LID + GHa mice exhibit a twofold increase in white adipose tissue mass, as well as increased levels of serum-free fatty acids and triglycerides, but no increase in the triglyceride content of liver and muscle. In conclusion, these results show that despite low levels of circulating IGF-1, insulin sensitivity in LID mice could be improved by inactivating GH action, suggesting that chronic elevation of GH levels plays a major role in insulin resistance. These results suggest that IGF-1 plays a role in maintaining a fine balance between GH and insulin to promote normal carbohydrate and lipid metabolism.

Recent epidemiological studies have suggested a statistical connection between serum IGF-1 levels in the upper quartile of the normal range and the relative risk of developing certain cancers. Our studies have focused on mouse models where circulating IGF-1 levels are reduced, while tissue expression of IGF-1 is normal. These mice show a lower risk for the development of colon and breast cancers and metastases when compared with control mice, and lend support to the hypothesis that circulating IGF-1 may be linked to cancer cell growth.

Female nonobese diabetic (NOD) mice develop spontaneous autoimmune sialadenitis and loss of salivary flow, and are a widely used model of Sjogren's syndrome. We examined the feasibility of local salivary gland immunomodulatory gene delivery to alter these sequelae in NOD mice. We constructed recombinant adeno-associated virus (rAAV) vectors encoding either human interleukin 10 (rAAVhIL-10) or beta-galactosidase (rAAVLacZ, control vector). Mice received rAAVhIL-10 or rAAVLacZ by retrograde submandibular ductal instillation either at age 8 weeks (early, before onset of sialadenitis), or at 16 weeks (late, after onset of sialadenitis). As a systemic treatment control, separate mice received intramuscular delivery of rAAVhIL-10 at each time point. Both submandibular and intramuscular delivery of vector led to low circulating levels of hIL-10. After submandibular administration of rAAVhIL-10, salivary flow rates at 20 weeks for both the early and late treatment groups were significantly higher than for both rAAVLacZ-administered and untreated mice. Systemic delivery of rAAVhIL-10 led to improved salivary flow in the late treatment group. Inflammatory infiltrates in submandibular glands, however, were significantly reduced only in mice receiving rAAVhIL-10 locally in the salivary gland compared with mice receiving this vector intramuscularly, or rAAVLacZ or no treatment. In addition, after submandibular rAAVhIL-10 delivery, NOD mice exhibited significantly lower blood glucose, and higher serum insulin, levels than all other groups, indicating some systemic benefit of this treatment. These studies show that expression of hIL-10 by rAAV vectors can have disease-modifying effects in the salivary glands of NOD mice, and suggest that local immunomodulatory gene transfer may be useful for managing the salivary gland pathology in Sjogren's syndrome.

Liver IGF-1 deficient (LID) mice demonstrate a 75% reduction in circulating IGF-1 levels and a corresponding fourfold increase in growth hormone (GH) levels. At 16 weeks of age, LID mice demonstrate, using the hyperinsulinemic-euglycemic clamp, insulin insensitivity in muscle, liver, and fat tissues. In contrast, mice with a gene deletion of the acid-labile subunit (ALSKO) demonstrate a 65% reduction in circulating IGF-1 levels, with normal GH levels and no signs of insulin resistance. To further clarify the relative roles of increased GH and decreased IGF-1 levels in the development of insulin resistance, we crossed the two mouse lines and created a double knockout mouse (LID+ALSKO). LID+ALSKO mice demonstrate a further reduction in circulating IGF-1 levels (85%) and a concomitant 10-fold increase in GH levels. Insulin tolerance tests showed an improvement in insulin responsiveness in the LID+ALSKO mice compared with controls; LID mice were very insulin insensitive. Surprisingly, insulin sensitivity, while improved in white adipose tissue and in muscle, was unchanged in the liver. The lack of improvement in liver insulin sensitivity may reflect the absence of IGF-1 receptors or increased triglyceride levels in the liver. The present study suggests that whereas GH plays a major role in inducing insulin resistance, IGF-1 may have a direct modulatory role.

Protein intake is a critical regulatory factor of the GH/IGF-I axis. Recently, it has been shown that splenic GH/IGF-I may respond to nutritional stress by preserving tissue homeostasis. To study the effects of exogenous administration of rhIGF-I on the splenic GH/IGF-I axis in protein malnourished rats, six-week-old male rats were assigned to one of four isocaloric diets differing in the protein content (0%, 4%, 12% and 20%) for a period of 12 days. Animals in the same dietary group on day 5 were randomly divided into two groups and during 7 days received a continuous subcutaneous infusion of either vehicle or rhIGF-I (300 microg/day). A low protein intake decreased the circulating levels of IGF-I, IGFBP-3, GH and insulin whereas the serum levels of IGFBP-1 were increased. Splenic IGFBP-3, -4 and -6 mRNA expression were up-regulated by protein malnutrition. Similarly, IGF-IR and GHR mRNA expression were significantly increased by the lack of dietary protein, whereas the levels of IGF-I mRNA remained unchanged. Exogenous rhIGF-I administration increased the circulating levels of IGFBP-1 and -3 in protein malnourished rats and reduced significantly the GH and insulin levels in well-fed rats. Similarly, rhIGF-I increased significantly the expression of the GHR in the spleen and splenic weight in all dietary groups, whereas nitrogen balance was enhanced only in the high-protein diet group. Among the cell subpopulations, B lymphocytes showed the highest GHR expression. These results suggest that in catabolic stress, induced by protein malnutrition the splenic GH/IGF-I axis is an important modulator and contributes to the maintenance of the homeostasis of the immune system.