Studies on function and regulation of the transcription factor C/EBPalpha

Abstract

The transcription factor C/EBPalpha was originally identified as a protein involved in hepatocyte-specific gene regulation. Later, it has been demonstrated that C/EBPalpha is expressed in several other tissues, mainly in adipose cells, lung, intestine, skin and in myeloid cells. C/EBPalpha takes part in cell-specific gene expression in these different cell types and has been suggested to be an important regulator of the terminal differentiation process, most clearly demonstrated in adipocytes. An antiproliferative effect of C/EBPalpha has also been observed in this cell type.

This thesis was aimed at further elucidation of the function of the C/EBPalpha protein, by using different rodent in vivo systems. Furthermore, the aim was to increase the knowledge about the transcriptional regulation of the C/EBPalpha gene itself. Our studies performed on the C/EBPalpha promoter by footprinting and gel-shift experiments, demonstrated 6 factor binding sites when an adult rat liver nuclear extract was used. Sites for USF and C/EBP were identified.

Transfection experiments showed that USF is the most important transactivator within the -350/+3 promoter fragment and that both C/EBPalpha itself and C/EBPbeta are able to transactivate this promoter. Expression studies of C/EBPalpha both during rat liver regeneration, after partial hepatectomy, and during chemically induced rat liver carcinogenesis, demonstrated a decreased expression of the C/EBPalpha gene attranscriptional, mRNA and protein levels . The observation that C/EBPalpha expression decreases during hepatocyte proliferation is in line with the previously suggested antimitotic function of the protein.

To obtain more information about the importance of C/EBPalpha in vivo we performed an inactivation of the gene in mice by homologous recombination in ES cells. Mice deficient in the C/EBPalpha protein show normal embryonic development, but dramatic phenotypical effects appear soon after birth. Thus, although apparently normal at birth, C/EBPalpha knockout mice die 7-10 hours postpartum, probably from hypoglycemia, since blood glucose levels are extremely low at the time of death. Histological analysis revealed very low levels of stored glycogen in the liver and low levels of fat in adipose tissues and liver in newborn knockout mice. These results suggest that C/EBPalpha is crucial for the regulation of genes involved in perinatal glycogen and fat synthesis in liver and adipose tissues.

Histological analysis of lungs from C/EBPalpha knockout mice, demonstrated a retarded pulmonary development and hyperproliferation of alveolar type II cells. Interestingly, about 20% of the newborn knockout mice showed severe respiratory problems, often resulting in death just some minutes after birth. The fact that type II cells are producers of surfactants, lipoproteins of importance in the alveolar inflation process, and that there is a known temporal correlation between C/EBPalpha expression and surfactant production, makes it possible that the observed respiratory dysfunction is due to inadequate surfactant levels in some of the C/EBPalpha knockout mice. Northern analysis of liver RNA demonstrated a decrease in albumin and an increase in a-fetoprotein mRNA levels. The levels of b-actin, c-jun and c-myc mRNA were all increased in livers from knockout mice. Furthermore, a 5-10 times increase in the frequency of hepatocytes producing the S-phase associated PCNA protein was observed.

Taken together, these results clearly indicate a less differentiated status and an increased proliferative activityin hepatocytes deficient in the C/EBPalpha protein. In conclusion, the results presented in this thesis, suggest that C/EBPalpha is an important factor in vivo for the acquisition of crucial differentiated functions in liver, adipose tissues and probably also in the lung. Furthermore, an antiproliferative role in hepatocytes is suggested.