NH2-terminal amino acid sequence of rabbit hepatopoietin A, a heparin-binding polypeptide growth factor for hepatocytes

Hepatopoietin A (HPTA) is an acidic heparin-binding polypeptide growth factor for hepatocytes with properties distinct from other known heparin-binding growth factors. HPTA is a heterodimer consisting of a heavy and a light polypeptide chain with Mr of 70,000 and 35,000 respectively. HPTA is a complete mitogen for hepatocytes in that it stimulates DNA synthesis in hepatocytes maintained in serum-free medium. Its complete purification from rabbit serum or human plasma was reported by us elsewhere (R. Zarnegar and G. Michalopoulos, 1989). In the present communication we report the N-terminal amino acid sequence of the HPTA light chain up to 24 residues (VVNGKPTRTNVGRMVSLKYRNKHI) and show that this sequence is unique and not related to any other proteins or growth factors based on computer search analysis. We have also raised antiserum against a synthetic peptide corresponding to the sequence of N-terminal amino acids residues 1 to 24, which recognizes the whole HPTA molecule. This antiserum as well as oligonucleotide probes corresponding to the N-terminal amino acid sequence of HPTA can be used as probes to identify tissue(s) of origin of this growth factor and assist in molecular cloning of its gene.     

Molecular analysis of gene deletion in aniridia-Wilms tumor association

Summary Hybrid clones were produced from the fusion of Chinese hamster cells and human fibroblasts from a patient with the aniridia-Wilms tumor association (AWTA). The DNA from the parental cells and the hybrid clones was screened by Southern blot and DNA hybridization with probes for the human insulin and Ha-ras-1 genes. Two alleles for the Ha-ras-1 gene were shown to exist in the AWTA cells by restriction fragment length polymorphism. One hybrid clone, containing a single allele for Ha-ras-1 was shown to contain a single chromosome 11 with a cytogenetically visible deletion at 11p13. The DNA from this hybrid contained the human genes for insulin, ^A, ^G, Ha-ras-1, and calcitonin, but lacked any human sequences homologous to a human catalase cDNA. This clone was also shown to express human lactate dehydrogenase A (LDH A) activity. These data indicate that the deletion of the affected chromosome in this AWTA patient begins distal to LDH A and includes band 11p13, but does not extend to calcitonin or other genes thought to be located in the distal half of chromosome 11p.     

Maintenance of microsomal cytochromes b5 and P-450 in primary cultures of parenchymal liver cells on collagen membranes

In previous reports from various laboratories, the levels of the microsomal cytochromes b₅ and P-450 in hepatocytes in primary culture have been found to be very low and difficult to measure. The studies reported in this paper demonstrate that cytochromes b₅ and P-450 in hepatocytes cultured on floating collagen membranes for periods of at least 10 days are maintained at levels readily measured by conventional techniques and comparable to those of liver $\text{in}$$\text{vivo}$. Addition of high levels of hydrocortisone (10^?4M) to the culture medium for periods up to 10 days resulted in further increases in the levels of these cytochromes. Cells cultured in the presence of hydrocortisone exhibited the appearance of cytochrome P-448, in contrast to the cells cultured in the absence of hydrocortisone, where cytochrome P-450 was maintained.     

Norepinephrine decreases EGF binding in primary rat hepatocyte cultures

Norepinephrine (NE) produced a dose-dependent inhibition of 125I-epidermal growth factor (EGF) binding to adult rat hepatocytes in primary culture. This effect was maximal after 1 hr of incubation with NE and could be blocked by the presence of an alpha 1-specific adrenergic receptor antagonist. The inhibition of binding correlates with the ability of NE to enhance hepatocyte DNA synthesis in the presence of EGF and appears to be mediated by a reduction in EGF receptor number, without a significant change in receptor affinity.     

Distinct morphological and mito-inhibitory effects induced by TGF-β1, HGF and EGF on mouse,rat and human hepatocytes

TGF-1 is known as a potent inhibitor of proliferation of rat and human hepatocytes. In this study we show that the effects of TGF-1 are quite different on mouse hepatocytes. In rat and human hepatocytes, TGF-1 inhibited DNA synthesis and also inhibited the morphological changes induced by growth factors in rat and human hepatocytes. In contrast, addition of TGF-1 to mouse hepatocytes resulted in pronounced alterations in morphology of these cells. These changes were similar to those induced by HGF and EGF. The induction of structural changes by TGF-1 was noted only in mouse hepatocytes. Mouse hepatocytes were also much more resistant to the mito-inhibitory effect of TGF-1. These findings suggest profound differences in hepatocyte growth regulation between these species and may relate to observed differences in susceptibility to carcinogenesis.Abbreviations EGF epidermal growth factor - HGF hepatocyte growth factor - SF scatter factor - TGF-1 transforming growth factor beta type one     

Acidic fibroblast growth factor (HBGF-1) stimulates DNA synthesis in primary rat hepatocyte cultures.

Acidic fibroblast growth factor (aFGF) stimulated DNA synthesis in primary rat hepatocyte cultures in a dose-dependent manner with maximal effect at 10-50 ng ml-1. This activity was dependent on the presence of heparin at a concentration of 10-50 micrograms.ml-1. Insulin interacted synergistically with aFGF, as it did with epidermal growth factor (EGF). The response to aFGF was only 50% that found with EGF. The disparity was not due to different kinetics of DNA synthesis, since the peak response for both growth factors occurred at 36-72 hr after plating of the hepatocytes. The potential relevance of this novel hepatocyte mitogen to normal and pathological liver growth is discussed.     

Differential effect of growth factors on growth stimulation and phenotypic stability of glutamine-synthetase-positive and -negative hepatocytes in primary culture

In rat liver parenchyma, two subpopulations of hepatocytes can be distinguished by the absence or presence of the marker enzyme, glutamine synthetase (GS). Hepatocytes in the perivenous zone immediately adjacent to the hepatic venules in the liver acinus are positive for GS. Using autoradiography in combination with immunocytochemistry, the response of these two hepatocyte populations (GS positive and GS negative) to a variety of growth factors (defined compounds or complex stimuli) was investigated in vitro. Irrespective of the individual growth-promoting activity (which varied considerably), all stimuli led to much higher labeling indices in GS-negative cells as compared to GS-positive cells. In GS-negative cells, the strongest effect was exerted by serum obtained from partially hepatectomized rats (labeling index, 67%) and the conditioned media of JM1 and JM2 hepatoma cells (63%-82%), followed by a combination of insulin and either norepinephrine (46%) or epidermal growth factor (EGF; 42%). In contrast, serum had the weakest influence on GS-positive cells (0.3%), while the other potent stimuli enhanced the labeling index of these cells by between 6% and 15% within 48 h. The percentage of labeled nuclei was higher in mononucleated than in binucleated GS-positive hepatocytes. The time course of thymidine incorporation was also different for the two subpopulations. Under all growth-promoting conditions, the stimulation of GS-negative cells peaked between 72 and 96 h, while it increased continuously in GS-positive cells for at least 120 h, particularly in the case of serum. In proliferating cultures, both the absolute and the relative number of GS-positive hepatocytes decreased, while no such effect was found in various nonproliferating control cultures maintained at low and high cell density. Similar results were found for GS activity. In contrast, the hormonal induction of tyrosine aminotransferase (TAT) was not affected. It is suggested that these differences in the growth response of GS-positive and -negative cells contribute to the acinar gradient in hepatocyte proliferation that occurs during liver regeneration. Furthermore, the striking phenotypic instability of GS-positive cells that have undergone DNA synthesis and mitosis supports the hypothesis that cellular reprogramming depends on passage through the cell cycle.     

Differential mitogenic effects of single chain hepatocyte growth factor (HGF)/scatter factor and HGF/NK1 following cleavage by factor Xa

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that is involved in many normal as well as pathological conditions. HGF/NK1, a splice variant of HGF/SF, has been reported to have either antagonistic or agonistic effects with regard to c-Met signaling depending on the cell type. In these experiments, we have determined that HGF/NK1 is a potent mitogen for rat hepatocytes in culture. Furthermore, we have found that coagulation factor Xa (fXa) is capable of cleaving HGF/NK1 and single chain HGF/SF (scHGF/SF). The products resulting from cleavage of HGF/NK1 or scHGF/SF by fXa appear as single bands under non-reducing conditions. The reaction products from the digestion of HGF/NK1 by fXa were separated under reducing conditions, and the cleavage site, as determined by N-terminal sequencing, was located C-terminal to arginine 134. Previous work established that the heparin-binding domain for HGF/SF is located in the N domain of HGF/SF. Additionally, the dimerization of the HGF/SF receptor (c-Met) by the ligand HGF/NK1 is facilitated by heparin and related sulfonated sugars on the cell surface, whereas heparin is not required for HGF/SF-mediated dimerization. Cleavage of single chain HGF/SF or HGF/NK1 by factor Xa does not alter the affinity of the respective molecules for heparin, but it did variably affect the associated mitogenic activity of these factors. The associated mitogenic activity of HGF/NK1 was reduced by more than 90%, whereas the mitogenic activity of scHGF/SF was unaffected. This suggests mandatory maintenance of a steric interaction of the N domain and the first kringle domain for HGF/NK1 to act as an agonist for rat hepatocyte growth but is not required by full-length HGF/SF.