A new member of a hepatoma-derived growth factor gene family can translocate to the nucleus

Hepatoma-derived growth factor (HDGF) and HDGF-related proteins (HRP) belong to a gene family with a well-conserved amino acid sequence at the N-terminus (the hath region). A new member of the HDGF family in humans and mice was identified and cloned; we call it HRP-3. The deduced amino acid sequence from HRP-3 cDNA contained 203 amino acids without a signal peptide for secretion. HRP-3 has its 97-amino-acid sequence at the N-terminus, which is highly conserved with the hath region of the HDGF family proteins. It also has a putative bipartite nuclear localizing signal (NLS) sequence in a similar location in its self-specific region of HDGF and HRP-1. Northern blot analysis shows that HRP-3 is expressed predominantly in the testis and brain, to an intermediate extent in the heart, and to a slight extent in the ovaries, kidneys, spleen, and liver in humans. Transfection of green fluorescent protein (GFP)-tagged HRP-3 cDNA showed that HRP-3 translocated to the nucleus of 293 cells. GFP-HRP-3 transfectants significantly increased their DNA synthesis more than cells transfected with vector only. The HRP-3 gene was mapped to chromosome 15, region q25 by FISH analysis. These findings suggest that a new member of the HDGF gene family, HRP-3, may function mainly in the nucleus of the brain, testis, and heart, probably for cell proliferation.     

Histone gene expression during sea urchin spermatogenesis: an in situ hybridization study

The expression of testis-specific and adult somatic histone genes in sea urchin testis was investigated by in situ hybridization. The testis-specific histone genes (Sp H2B-1 of Strongylocentrotus purpuratus and Sp H2B-2 of Lytechinus pictus) were expressed exclusively in a subset of male germ line cells. These cells are morphologically identical to replicating cells pulse-labelled with 3H-thymidine. Genes coding for histones expressed in adult somatic and late embryo cells (H2A-beta for S. purpuratus and H3-1 for L. pictus) were expressed in the same germ line cells, as well as in the supportive cells (nutritive phagocytes) of the gonad. All histone mRNAs detected in the male germ lineage declined precipitously by the early spermatid stage, before cytoplasmic reduction. The data suggest that both testis-specific and adult somatic histone genes are expressed in proliferating male germ line cells. Testis-specific gene expression is restricted to spermatogonia and premeiotic spermatids, but somatic histone expression is not. The decline of histone mRNA in nondividing spermatids is not merely a consequence of cytoplasmic shedding, but probably reflects mRNA turnover.     

Up-Regulation of Hepatoma-Derived Growth Factor Facilities Tumor Progression in Malignant Melanoma

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200) showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn) expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16–F10 melanoma cells whereas HDGF knockdown exerted opposite effects in vitro. To evaluate the effects of HDGF on tumour growth and metastasis in vivo, syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.     

一个新的睾丸特异表达基因的克隆及功能的初步分析

运用“数据库消减杂交”(digital differential display)方法来筛选人类睾丸特异表达新基因,获得了有差异显示的代表新基因的克隆重叠群。挑选其中一个克隆重叠群HS．326528进行多组织RT—PCR,初步获得该重叠群在人睾丸中有高表达。从该重叠群的IMAGE出发,采用生物信息学的方法快速克隆了一个人类新基因的全长cDNA序列,其全长1044bp,开放阅读框为214～529bp,定位于15q26．2,编码由105个氨基酸组成、分子量为11．7kD、等电点为10．09的一个碱性蛋白,该蛋白与已知蛋白无明显的同源性,克隆实验证明该基因的阅读框完全正确,RT—PCR和Northern blot显示该基因在人类睾丸中特异表达,实时PCR结果表明：该基因在成人睾丸中高表达,在精子中有中度表达,在胚胎睾丸中低表达,推测该基因与精子的生成有关,命名为SRG8(homo sapiens spermatogenesis—related gene 8)(GenBank登录号：AY489187),该基因编码的蛋白定位于细胞核。流式结果分析表明,SRG8基因能够促使HeLa细胞由S期向G2期的转变,从而加速细胞的分裂。这些结果表明SRG8基因可能在睾丸的发育及精子的形成过程中起重要的作用。     

Identification of a novel testis-specific gene mtLR1, which is expressed at specific stages of mouse spermatogenesis

A novel testis-specific gene termed mtLR1 was identified by digital differential display. Sequence analyses revealed that mtLR1 protein contains an amino terminus leucine-rich repeat domain and shows 33% similarities to PIDD which functions in p53-mediated apoptosis. Northern blot analysis showed that mtLR1 mRNA was specifically expressed in adult mouse testis, and RT-PCR results also showed that mtLR1 was exclusively expressed in adult testis and not in spermatogonial cells. The expression of mtLR1 mRNA was developmentally upregulated in the testes during sexual maturation and was, conversely, downregulated by experimental cryptorchidism in vivo. We also showed that the expression of mtLR1 mRNA was relatively highly sensitive to heat stress in vitro. The green fluorescent protein produced by pEGFP-C3/mtLR1 was only detected in the cytoplasm of spermatogonia cell line GC-1 after 24 h posttransfection. Immunohistochemical analysis revealed that the protein is most abundant in the cytoplasm of spermatocytes and round spermatids within seminiferous tubules of the adult testis. The time-dependent expression pattern of mtLR1 in postnatal mouse testes suggested that mtLR1 gene might be involved in the regulation of spermatogenesis and sperm maturation.     

Interaction of HRP-2 isoforms with HDGF: chromatin binding of a specific heteromer

Hepatoma-derived growth-factor-related protein 2 (HRP-2) belongs to a family with five additional members: hepatoma-derived growth factor (HDGF); lens epithelium-derived growth factor; and the HDGF-related proteins -1, -3 and -4. Very little is known regarding the function of HRP-2 in particular. This study shows for the first time heteromer formation of different members of the HRP family; HDGF and HRP-2. In addition, we discovered a previously unknown splice variant of HRP-2 mRNA encoding for a protein with a 53-amino acid deletion in its hath region. This HRP-2 isoform c interacts preferentially with a processed form of HDGF probably because of the loss of an α helix of HRP-2. Furthermore, in contrast to other isoforms of HRP-2, isoform c binds to chromatin similar to its most closely related family member lens epithelium-derived growth factor with potential consequences regarding its function in HIV integration. Interestingly, only the new HRP-2 isoform c and a processed form of HDGF are displaced from condensed mitotic metaphase chromatin. In conclusion, these observations provide a new perspective for understanding the biological functions of HDGF and related proteins.     

Hepatoma-derived growth factor, a new trophic factor for motor neurons, is up-regulated in the spinal cord of PQBP-1 transgenic mice before onset of degeneration

Hepatoma-derived growth factor (HDGF) is a nuclear protein homologous to the high-mobility group B1 family of proteins. It is known to be released from cells and to act as a trophic factor for dividing cells. In this study HDGF was increased in spinal motor neurons of a mouse model of motor neuron degeneration, polyglutamine-tract-binding protein-1 (PQBP-1) transgenic mice, before onset of degeneration. HDGF promoted neurite extension and survival of spinal motor neurons in primary culture. HDGF repressed cell death of motor neurons after facial nerve section in newborn rats in vivo. We also found a significant increase in p53 in spinal motor neurons of the transgenic mice. p53 bound to a sequence in the upstream of the HDGF gene in a gel mobility shift assay, and promoted gene expression through the cis-element in chloramphenicol acetyl transfer (CAT) assay. Finally, we found that HDGF was increased in CSF of PQBP-1 transgenic mice. Collectively, our results show that HDGF is a novel trophic factor for motor neurons and suggest that it might play a protective role against motor neuron degeneration in PQBP-1 transgenic mice.     

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.Neuritogenesis is a key step in nervous system development in which neurons extend dendrites and axons and connect to different targets in and outside the nervous system. The proper regulation of this process is controlled by a number of extra- and intracellular molecules expressed by neurons themselves or non-neuronal cells in their surroundings. Multiple studies indicate that rearrangement of the neuronal cytoskeleton in response to extracellular signals is an important mechanism during neurite extension and pathfinding (1-3). Manipulation of the polymerization and depolymerization of microtubules has shown that regulation of microtubule assembly and maintenance is important for neuritogenesis (4). Microtubule dynamics are regulated by a huge number of regulatory proteins like tau or other microtubule-associated proteins (MAPs)⁴ (5). In addition, proteins like CRMP-2 that interact with tubulin dimers and accelerate the assembly of tubulin into microtubles have been shown to be involved in the regulation of neuronal polarity and neuritogenesis (6-10). Despite all advances, however, made in the understanding of the role of the cytoskeleton and its regulatory proteins during neuritic growth there are still many open questions regarding the regulation of these processes. Therefore identifying new molecules binding to and modulating the turnover of microtubules is of high interest for the understanding of how neurite outgrowth is regulated.Hepatoma-derived growth factor (HDGF) is a protein that was purified from secretions of hepatoma cells by virtue of its growth factor activity. Subsequently 5 additional proteins were identified in which the 97 N-terminal amino acid residues show strong similarity to HDGF. Accordingly this family of proteins has been termed HDGF-related proteins (HRP) (11-13). HDGF has neurotrophic activity for hippocampal, spinal, and facial motor neurons (14, 15). So far, however, no receptor or signal transduction pathway involved has been identified for any of the HRPs.Most HRPs are expressed in a variety of tissues. HRP-3, however, is the only family member in whose expression is restricted. It is only expressed in neurons and to a low extent in glial cells (16, 17). Like HDGF after transfection into human embryonic kidney cells HRP-3 exhibits proliferative activity (12). The strong and almost exclusive expression of HRP-3 in postmitotic neurons, however, suggests biological functions other than its growth factor activity (16).In the present study we examine the expression and function of HRP-3 protein during mouse embryonic neuronal development. We demonstrate that the protein locates to the cytoplasm and neurites during early nervous system development, whereas most of HRP-3 can be found in the nucleus in adult neurons. We show that HRP-3 promotes neurite growth and suggest that this is due to the interaction of HRP-3 with the tubulin component of the neuronal cytoskeleton.