Changes in Hepatocyte Nuclear Factor 4 (HNF-4) during Liver Failure: Understanding the Alpha (a) and the Gamma (g) Forms

Performing sundry functions that have effects on nearly every other system of the human body, the liver arises as truly a most vital and remarkably complex organ, with respect to the functions it undertakes. Since the liver is such an essential organ in the human body, if damaged, one's very subsistence can be placed in jeopardy.

Among the liver's most significant functions are (1) the regulation of the body's metabolism and (2) the synthesis of blood proteins that each hold a specific mechanistic function in the human body. With respect to metabolism, the liver stores excess glucose (sugar), the main form of energy for bodily processes to occur, in the form of glycogen that can be converted back to glucose when blood-glucose levels are low. The liver regulates the conversion of toxic ammonia into less toxic urea for filtration and excretion by the kidneys. With respect to the synthesis of blood proteins, the liver manufactures blood proteins such as albumin, which regulates the movement of water in body tissues, and fibrinogen, which is an instrumental clotting factor in healing bodily wounds. The key question that naturally abounds is the following: how does the liver regulate metabolism and manufacture necessary blood proteins?

It is known that the liver performs all the processes of secretion, regulation and storage via the use of specific genes for each operation that are located on the chromosomes inside the nucleus of hepatocytes. For each specific function that the liver carries out, genes are involved in encoding specific enzymes for these specific functions. What induces genes to activate are the liver's liver-specific transcription factors, proteins that signal RNA polymerase to transcribe the gene into mRNA. Such liver-specific transcription factors, hepatocyte nuclear factor 4 (HNF-4) for example, are so integral to the liver's operation that without them, the liver would cease to function.

Dr. Ponder's laboratory has been studying a model for liver failure in which lipopolysaccharide (LPS) is the singular agent in causing liver failure. In rats that were induced with 5mg/kg LPS, there was a moderate decrease in liver functions; however, no rats died. In rats that were induced with 50mg/kg LPS, there was a grave decrease in liver functions and deaths resulted. Therefore, a logical hypothesis emerged as follows: (1) A decrease in HNF-4 may contribute to liver failure after LPS treatment, and (2) a higher dose of LPS will cause a severe decrease in HNF-4. In both doses the rats experienced a complete loss of the major protein.

There are two related genes encoding the HNF-4 protein, HNF-4a and HNF-4g. Each has a high homology to one another and each encodes different proteins that contain differing sequences due to alternative splicing of mRNA. In performing western blots to assess the effect of LPS on HNF-4 protein levels (at both 5mg/kg and 50mg/kg over a 24-hour period), it was found that a 50kD band appeared. This 50kD band contains the middle region epitope of HNF-4a, which is greatly conserved. Therefore, one hypothesis made was that this 50kD band was either an unusual HNF-4a or HNF-4g . Although HNF-4g is absent in normal mouse liver, it might be induced under certain conditions, and its size is known to be 50kD. Therefore, it was predicted that the 50kD protein band in the rat LPS model would be HNF-4g. A further hypothesis that we constructed was that since this 50kD band was appearing when levels of HNF-4a waned, this 50kD band (HNF-4?) could be replacing the function of the missing HNF-4a.

Therefore, my project involved sequencing the full length of the HNF-4g cDNA, which has not been fully studied. In my project, we made the assumption that we were in fact dealing with the HNF-4g. Subsequently, we tested livers for g-RNA and used the RNA in RT-PCR in order to amplify specific sequences of mouse HNF-4g. The PCR product was cloned into a retroviral vector, purified, and then sequenced. The resulting sequence we obtain from this project will be used in further answering the following central questions: What is this 50kD band? Might it be HNF-4g?