Mekky M. Abouzied

Expression patterns and different subcellular localization of the growth factors HDGF (hepatoma-derived growth factor) and HRP-3 (HDGF-related protein-3) suggest functions in addition to their mitogenic activity.

HDGF (hepatoma-derived growth factor) and the HRPs (HDGF-related proteins) comprise a family of six proteins which display high identity in their N-terminus, but differ at the C-terminus. Here we investigate the patterns of expression of HDGF and HRP-3, by generating antisera specifically recognizing each growth factor. Whereas HRP-3 protein is expressed only in brain, HDGF can be found in a broad range of tissues, with highest levels in brain, testis, lung and spleen. The expression of HDGF and HRP-3 was found to be regulated during brain development, with highest levels around birth, followed by a decline until postnatal day 9. Interestingly, expression of HRP-3 increases again in adult brain. In situ hybridization and immunohistochemistry of cerebellar, cerebral and hippocampal brain slices showed that expression of both growth factors is not limited to areas of high proliferative activity. Both mRNAs and proteins are expressed in neuronal as well as glial cells. Immunocytochemistry of cultured neocortical neurons revealed that HDGF and HRP-3 can be found in the nucleus as well as the cytoplasm. HDGF is restricted to the neuronal soma, whereas HRP-3 can also be found in neurites. Thus the expression of HDGF and HRP-3 in differentiated cells, post-mitotic neurons and primary cultures of rat neocortex points to functions in brain that might not be limited to proliferation. In addition, their simultaneous expression in the same cell and their different subcellular localization in cultured neurons suggest different functions of HDGF and HRP-3 within single cells.

Expression of Hepatoma-derived growth factor family members in the adult central nervous system

BackgroundHepatoma-derived growth factor (HDGF) belongs to a polypeptide family containing five additional members called HDGF related proteins 1–4 (HRP-1 to -4) and Lens epithelial derived growth factor. Whereas some family members such as HDGF and HRP-2 are expressed in a wide range of tissues, the expression of others is very restricted. HRP-1 and -4 are only expressed in testis, HRP-3 only in the nervous system. Here we investigated the expression of HDGF, HRP-2 and HRP-3 in the central nervous system of adult mice on the cellular level by immunohistochemistry. In addition we performed Western blot analysis of various brain regions as well as neuronal and glial cell cultures.ResultsHDGF was rather evenly expressed throughout all brain regions tested with the lowest expression in the substantia nigra. HRP-2 was strongly expressed in the thalamus, prefrontal and parietal cortex, neurohypophysis, and the cerebellum, HRP-3 in the bulbus olfactorius, piriform cortex and amygdala complex. HDGF and HRP-2 were found to be expressed by neurons, astrocytes and oligodendrocytes. In contrast, strong expression of HRP-3 in the adult nervous system is restricted to neurons, except for very weak expression in oligodendrocytes in the brain stem. Although the majority of neurons are HRP-3 positive, some like cerebellar granule cells are negative.ConclusionThe coexpression of HDGF and HRP-2 in glia and neurons as well as the coexpression of all three proteins in many neurons suggests different functions of members of the HDGF protein family in cells of the central nervous system that might include proliferation as well as cell survival. In addition the restricted expression of HRP-3 point to a special function of this family member for neuronal cells.

Hepatoma‐derived growth factor (HDGF) is dispensable for normal mouse development

Hepatoma‐derived growth factor (HDGF) is suggested to be involved in organ development and exhibits proliferative, angiogenic, and neurotrophic activity. The in vivo functions are, however, so far unknown. In this study, we generated HDGF‐deficient mice, in which parts of the HDGF gene were replaced by a gene encoding green fluorescent protein (eGFP). HDGF−/− mice are viable with no apparent morphological abnormalities. Cultured HDGF‐deficient dermal fibroblasts show unaltered proliferation rates and cell‐cycle distributions. In contrast to previous studies, our data demonstrate that signal pathways involved in the response to extracellular HDGF do not depend on the presence of intracellular HDGF. Contrary to the reported role of HDGF as a modulator of apoptosis, similar apoptotic rates were found between wild‐type and HDGF‐deficient fibroblasts following tumor necrosis factor α (TNFα) ‐induced apoptosis or cellular stress. The lack of obvious biochemical and morphological phenotypes in HDGF‐deficient mice demonstrates that in vivo HDGF is dispensable for normal development in mice. Developmental Dynamics 237:1875–1885, 2008.

Hepatoma-derived Growth Factor-related Protein-3 Interacts with Microtubules and Promotes Neurite Outgrowth in Mouse Cortical Neurons

Hepatoma-derived growth factor-related proteins (HRP) comprise a family of 6 members, which the biological functions are still largely unclear. Here we show that during embryogenesis HRP-3 is strongly expressed in the developing nervous system. At early stages of development HRP-3 is located in the cytoplasm and neurites of cortical neurons. Upon maturation HRP-3 relocalizes continuously to the nuclei and in the majority of neurons of adult mice it is located exclusively in the nucleus. This redistribution from neurites to nuclei is also found in embryonic cortical neurons maturing in cell culture. We show that HRP-3 is necessary for proper neurite outgrowth in primary cortical neurons. To identify possible mechanisms of how HRP-3 modulate neuritogenesis we isolated HRP-3 interaction partners and demonstrate that it binds tubulin through the N-terminal so called HATH region, which is strongly conserved among members of the HRP family. It promotes tubulin polymerization, stabilizes and bundles microtubules. This activity depends on the extranuclear localization of HRP-3. HRP-3 thus could play an important role during neuronal development by its modulation of the neuronal cytoskeleton.

Overexpression of hepatoma-derived growth factor in melanocytes does not lead to oncogenic transformation

BackgroundHDGF is a growth factor which is overexpressed in a wide range of tumors. Importantly, expression levels were identified as a prognostic marker in some types of cancer such as melanoma.MethodsTo investigate the presumed oncogenic/transforming capacity of HDGF, we generated transgenic mice overexpressing HDGF in melanocytes. These mice were bred with mice heterozygous for a defective copy of the Ink4a tumor suppressor gene and were exposed to UV light to increase the risk for tumor development both genetically and physiochemically. Mice were analyzed by immunohistochemistry and Western blotting. Furthermore, primary melanocytes were isolated from different strains created.ResultsTransgenic animals overexpressed HDGF in hair follicle melanocytes. Interestingly, primary melanocytes isolated from transgenic animals were not able to differentiate in vitro whereas cells isolated from wild type and HDGF-deficient animals were. Although, HDGF-/-/Ink4a+/- mice displayed an increased number of epidermoid cysts after exposure to UV light, no melanomas or premelanocytic alterations could be detected in this mouse model.ConclusionsThe results therefore provide no evidence that HDGF has a transforming capacity in tumor development. Our results in combination with previous findings point to a possible role in cell differentiation and suggest that HDGF promotes tumor progression after secondary upregulation and may represent another protein fitting into the concept of non-oncogene addiction of tumor tissue.

Hepatoma‐derived growth factor‐related protein‐3: A new neurotrophic and neurite outgrowth‐promoting factor for cortical neurons

Hepatoma‐derived growth factor‐related proteins (HRPs) make up a family of six members. Hepatoma‐derived growth factor‐related protein‐3 (HRP‐3) is the only family member whose expression is almost restricted to nervous tissue. Here we show that soluble HRP‐3 acts as a novel neurotrophic factor for cultured primary cortical neurons. Antibody‐mediated neutralization of HRP‐3 function results in neuronal degeneration. In contrast, HRP‐3 as the only addition to a culture medium not supporting neuronal survival rescues neurons to an extent comparable to the addition of FCS. Besides this neuroprotective capability, the protein exerts a neurite outgrowth‐promoting effect when it is presented as a coated substrate but not as a soluble factor. This study points to an important role of HRP‐3 during the development of the nervous system.

Misfolded endoplasmic reticulum retained subunits cause degradation of wild-type subunits of arylsulfatase A heteromers

Arylsulfatase A is an oligomeric lysosomal enzyme. In the present study, we use this enzyme as a model protein to examine how heteromerization of wild‐type and misfolded endoplasmic reticulum‐degraded arylsulfatase A polypeptides affects the quality control of wild‐type arylsulfatase A subunits. Using a conformation sensitive monoclonal antibody, we show that, within heteromers of misfolded and wild‐type arylsulfatase A, the wild‐type subunits are not fully folded. The results obtained show that arylsulfatase A polypeptide complexes, rather than the monomers, are subject to endoplasmic reticulum quality control and that, within a heteromer, the misfolded subunit exerts a dominant negative effect on the wild‐type subunit. Although it has been shown that mature lysosomal arylsulfatase A forms dimers at neutral pH, the results obtained in the present study demonstrate that, in the early biosynthetic pathway, arylsulfatase A forms oligomers with more than two subunits.

Diagnostic utility of leptin and insulin-like growth factor binding protein-2 in hepatocellular carcinoma of diabetic and non-diabetic Egyptian patients

Purpose: To elucidate the possible diagnostic utility of adipokines and insulin growth factor binding proteins in hepatocellular carcinoma (HCC) diabetic subjects. Methods: Seventy five patients were divided equally into 3 groups as follows: healthy normal control (NC), non-diabetic hepatocellular carcinoma (HCC) and diabetic hepatocellular carcinoma (HCC-DM). Serum levels of leptin, insulin growth factor binding protein-2 (IGFBP-2) and alpha fetoprotein (AFP) were measured. Correlation and receiver operating characteristics (ROC) analysis was carried out. Results: HCC and HCC-DM groups showed changes in body mass index (BMI, p > 0.05 and p < 0.001 respectively), glucose, insulin, homeostatic model assessment-insulin resistance (HOMA-IR), liver function tests and AFP (p < 0.001). Leptin levels increased significantly in both HCC and HCC-DM (p < 0.001). Furthermore, IGFBP-2 showed significant increase in both groups (p < 0.001). Both leptin and insulin-like growth factor binding protein-2 (IGFBP-2) displayed significant positive correlation with AFP (p < 0.001). ROC analysis indicate different diagnostic accuracies for the tested markers for the various groups. Conclusion: Leptin and IGFBP-2 demonstrate significant potentials as diagnostic tools for HCC patients, especially diabetic cases, with IGFBP-2 displaying the highest diagnostic accuracy for HCC and HCC-DM groups. Keywords: Hepatocellular carcinoma, Diabetes mellitus, Leptin, Insulin-like growth factor-binding proteins-2, Adipokines