Keiichi Itakura, Ph.D.

Molecular Events in Energy Balance
During study of the mechanisms underlying activation of human cytomegalovirus (HCMV), we have cloned modulator recognition factor (Mrf )-1 and -2 that interact with the modulator region of the HCMV major immediate early gene. These proteins belong to the family of AT-rich interaction domain (ARID) proteins found in organisms as diverse as insects, yeast, mammals, and plants, and are thought to control gene expression in differentiation and development.

To investigate the functions of these proteins, we have generated transgenic mice that lack the ability to express Mrf-2 (Mrf-2KO). The most striking characteristic of Mrf-2KO is their extreme leanness. Indeed, this persists even when they are subjected to a high-fat diet for prolonged periods. They consume energy when at rest and metabolize glucose at an accelerated rate. To identify the origin of the lean body of Mrf-2KO mice, we have exploited an in vivo experimental system in which fibroblasts isolated from mouse embryos are induced to differentiate into fat cells by the application of a defined hormone mixture. We have shown that fat differentiation is severely impaired in cells that are derived from Mrf-2KO embryos in comparison to cells derived from their normal littermates. Examination of the expression of key genes involved in fat cell differentiation demonstrates that Mrf-2KO cells express significantly lower levels of genes than are required for the later stages. These results suggest that Mrf-2 is essential for expression of genes developing the ability to accumulate fat. This makes Mrf-2 an attractive target for the development of new therapeutic agents in the fight against obesity and diabetes.

To obtain preliminary data on the expression of Mrf-1and -2, we investigated a few cell lines under different conditions. These results show that expression of the Mrf-1 mRNA is induced by a treatment of growth factor (IGF-1, EGF), cytokine (IL-6), and adipokine (leptin) in prostate cells. On the other hand, that of the Mrf-2 mRNA is suppressed by the same treatment. Cell cycle analysis shows that expression of the Mrf-1 mRNA is induced toward S phase, and that of the Mrf-2 mRNA is highest at G0/G1 phase and reduced toward S phase. Furthermore, induction of the Mrf-1 mRNA and reduction of the Mrf-2 mRNA are observed under hypoxia conditions. These results suggest an antagonistic relationship between Mrf-1 and Mrf-2. We will further investigate the functions of these two proteins in differentiation and hypoxia.

Functions of Homeobox Genes in Carcinogenesis
We surveyed the expression of homeobox genes (HOX9-13) in four clusters, designated A, B, C, and D, in human tissues and found that the genes are mainly expressed in tissues present at posterior portions of the body (prostate and testis). Further examination of various cancer cells reveals that the HOXC-11 gene is expressed in all of the prostate cancer cell lines we tested. Therefore, we focus our research on investigating the functions of the HOXC-11 gene in cancer cells to study prostate carcinogenesis. Expression of the gene is higher in low-density cultures than in high-density cultures, with the lowest expression in confluent cultures.

Cell cycle analysis demonstrates that the gene is expressed in G1 phase and disappears in S phase. Interestingly, knockdown experiments of the HOXC-11 mRNA by siRNA induce the  expression of P21 in the prostate cancer cell line DU145. Forced expression of the HOXC-11 in DU145 gene stimulates cell proliferation in comparison to control cells. These results lead us to hypothesize that HOXC-11 could suppress the expression of P21 to stimulate cell proliferation.