Biological Activity And Its Mechanism Of A Novel Human Hepatic Growth Factor: Hepatopoietin Cn

The mammalian liver possesses an extraordinary capacity for responding to physical or chemical injuries by regenerating the capability of replenishing lost functions and mass. Liver regeneration has been confirmed to be a very complex process, and hundreds of substances participate in it. However, it was difficult to demonstrate that any of these factors itself had an essential or preferential role in liver regeneration.During the past decades, accumulative evidence has been collected on the existence of specific hepatic substance capable of stimulating hepatocyte proliferation and liver regeneration. In 1975 LaBrecque found the existence of a kind of hepatic stimulating substance (HSS) from weanling rat liver, which was able to stimulate hepatic DNA synthesis both in vitro and in vivo. Although during past 30 years, the physicochemical characteristics of HSS from mammals or human beings and the potential for treatment of hepatic diseases have been investigated, its essential nature in composition and structure remains puzzling and hurdle in further research and development, as its purified HSS has not been obtained so far.In recent years, we isolated a pure protein with hepatic stimulatory activity from the extract of weanling calf liver. We cloned its gene from human fetal liver cDNA library. Sequencing analysis showed that it is a member of the leucine-rich acidic nuclear protein family (the LANP family), and named as hepatopoietin Cn (HPPCn). The LANP family is involved in a variety of cellular processes in both nucleus and cytoplasm, including signaling, protein degradation, cytoskeletal dynamics, and morphogenesis. However, the extracellular activity has not been reported.HPPCn is a member of LANP family, containing two possible nuclear localization signals and lacking signal peptide. How can HPPCn be exported from cells and exert extracellular function. Evidences have been collected that some nuclear proteins (such as HMGB1 and HDGF) could be exported from cells in the absence of a functional ER/Golgi system, playing an essential role in proliferation, inflammation and differentiation.Here, with the availability of the HPPCn gene and its recombinant product, a series of questions about HPPCn has been addressed, such as expression profile, hepatic stimulating activity in vitro and in vivo, and action mechanisms.Firstly, in order to better understand the biological activity of HPPCn in liver regeneration, we studied the expression profile of HPPCn in different cells and tissues, and in different time after 34% partial hepatectomy. HPPCn appeared to be expressed in multiple cells and tissues, and more in brain, heart, liver and kidney. It was located to nuclei in SMMC7721 hepatoma cell line. The mRNA level of HPPCn in remnant mice liver rose shortly after PH, and peaked at 2hr and 72hr postoperatively, and the protein level of HPPCn increased slowly within 12hr after PH and declined thereafter, which showed that the level of HPPCn is influenced with time after partial hepatectomy, and supported that HPPCn plays a role in the process of liver regeneration.Secondly, the subsequent experiments were carried out to identify the stimulating activity in vitro and in vivo of recombinant product (rhHPPCn). A dose-dependent stimulation of the ³H-TdR incorporation into DNA has been detected in SMMC7721, L02 hepatic cell lines and primary cultured hepatocytes exposed to rhHPPCn, and showed the partial hepatic cell specificity similar to HSS on different cell lines. The proliferation activity was confirmed by MTT and CFSE-based proliferation assay. Additionally, injection of 2.5mg rhHPPCn/kg body weight yielded a dramatic increase of the ³H-TdR/BrdU incorporation into DNA in the remnant mice liver after 34% partial hepatectomy. And a moderate but statistically significant increase of hepatic DNA synthesis was found in normal liver, but had no effects in the kidney, cerebellum, and spleen. Injection of rhHPPCn to carbon tetrachloride treated mice reduced the enhanced level of serum GPT and GOT, and increased the expression of proliferating cell nuclear antigen (PCNA) in liver, indicating the stimulation to hepatic proliferation and protection from CCl₄ induced liver damage.Finally, we investigated the mechanism of HPPCn acting as a growth factor. HPPCn was secreted by SMMC7721 in physiological conditions. Besides, we showed that an endogenous HPPCn-suppression in SMMC7721 was induced by exogenously supplied rhHPPCn, which means that there is a feedback regulation inside and outside. And evidences were collected using binding assay on the existence of receptors on hepatic cells surface. The binding of HPPCn to receptors is specific, saturated, and reversible. The effect of rhHPPCn on different signal-transduction pathway related to cell proliferation was observed in vitro and in vivo. As a result, rhHPPCn can simultaneously activate SPK, Erk1/2 and Stat3 in SMMC7721 cell line in a dose-dependent manner. And rhHPPCn treatment resulted in a dramatic increase of Erk1/2 phosphorylation in remnant liver of PH mice. The growth stimulating activity of rhHPPCn was validated.In conclusion, we identified the extracellular activity of a novel leucine-rich acidic nuclear protein HPPCn as a growth factor. We suggest that HPPCn can not only stimulate the proliferation of primary cultured rat liver cells, SMMC7721 and L02 cell lines, but also can enhance the regeneration of hepatectomized liver, which is similar to that of HSS. And we suggested that HPPCn may activate intercellular signal-transduction pathway by interacting with its receptors on cells surface. Hence, the exploitation of HPPCn from HSS paves a way for clarifying the mechanism of liver regeneration and its potential on treatment of liver diseases.