Function Of Hepatocyte Growth Factor Research Articles

Hepatocyte growth factor (HGF) gene: molecular characterisation of complete coding sequence and expression profile in Tarim red deer (Cervus hanglu yarkandensis) antlers

Context The cDNA sequence of hepatocyte growth factor (HGF) gene in Tarim red deer has not been reported yet. Aims This study aims to obtain the full-length cDNA sequence of HGF and analyse its expression in different parts of developing antler tissues. The result provides foundational data for understanding the potential role of HGF in regulating antler growth and development. Methods Rapid amplification of cDNA ends was used to obtain the full-length cDNA sequence of Tarim red deer HGF. The pET28a (+) vector was constructed for prokaryotic expression of the recombinant protein in E. coli BL21 (DE3). The expression of HGF in different antler tissues was detected using real-time quantitative polymerase chain reaction and immunohistochemistry. Key results The full-length cDNA of Tarim red deer HGF was found to consist of a 156 bp 5’untranslated region (UTR), a 112 bp 3’UTR, and a 2193 bp open reading frame encoding a protein of 730 amino acids. The recombinant HGF protein expressed in prokaryotic cells formed inclusion bodies. HGF and its receptor c-Met were expressed in all four different antler tissues, with the highest expression level in velvet skin, followed by bone, cartilage, and the lowest in the mesenchyme. Conclusions The study successfully obtained the full-length cDNA sequence of Tarim red deer HGF and identified the expression profile of HGF and c-Met in different antler tissues. HGF is a candidate gene that may play a role in regulating the growth and development of deer antler. Implications These findings provide fundamental data for further investigations into the role of HGF in antler development. Understanding the function of HGF in antler development could have implications for elucidating the mechanism of antler regeneration.

Hepatocyte growth factor is necessary for efficient outgrowth of injured peripheral axons in in vitro culture system and in vivo nerve crush mouse model

Hepatocyte growth factor (HGF) is a neurotrophic factor and its role in peripheral nerves has been relatively unknown. In this study, biological functions of HGF and its receptor c-met have been investigated in the context of regeneration of damaged peripheral nerves. Axotomy of the peripheral branch of sensory neurons from embryonic dorsal root ganglia (DRG) resulted in the increased protein levels of HGF and phosphorylated c-met. When the neuronal cultures were treated with a pharmacological inhibitor of c-met, PHA665752, the length of axotomy-induced outgrowth of neurite was significantly reduced. On the other hand, the addition of recombinant HGF proteins to the neuronal culture facilitated axon outgrowth. In the nerve crush mouse model, the protein level of HGF was increased around the injury site by almost 5.5-fold at 24 h post injury compared to control mice and was maintained at elevated levels for another 6 days. The amount of phosphorylated c-met receptor in sciatic nerve was also observed to be higher than control mice. When PHA665752 was locally applied to the injury site of sciatic nerve, axon outgrowth and injury mediated induction of cJun protein were effectively inhibited, indicating the functional involvement of HGF/c-met pathway in the nerve regeneration process. When extra HGF was exogenously provided by intramuscular injection of plasmid DNA expressing HGF, axon outgrowth from damaged sciatic nerve and cJun expression level were enhanced. Taken together, these results suggested that HGF/c-met pathway plays important roles in axon outgrowth by directly interacting with sensory neurons and thus HGF might be a useful tool for developing therapeutics for peripheral neuropathy.

Inhibition of hepatocyte growth factor/c-Met signalling abrogates joint destruction by suppressing monocyte migration in rheumatoid arthritis

To determine the expression of hepatocyte growth factor (HGF) in RA biological fluids, the role of HGF in monocyte migration and the therapeutic effect of the c-Met inhibitor savolitinib in an arthritis model mice. HGF/c-Met expression in serum, SF and synovial tissues (STs) obtained from RA patients and controls, as well as RA fibroblast-like synoviocytes (FLSs), was evaluated by ELISA and immunostaining. To determine the function of HGF in RA SF, we preincubated RA SF with a neutralizing anti-HGF antibody and measured the chemotactic ability of a human acute monocytic leukaemia cell line (THP-1). Additionally, examinations were conducted of SKG mice treated with savolitinib for 4 weeks. HGF levels in serum from RA patients were significantly higher than those in the controls and were decreased by drug treatment for 24 weeks. Additionally, the HGF level in SF from RA patients was higher than that in SF from OA patients. HGF and c-Met expression was also noted in RA STs. Stimulation of RA FLSs with TNF-α increased HGF/c-Met expression in a concentration-dependent manner, and c-Met signal inhibition suppressed production of fractalkine/CX3CL1 and macrophage inflammatory protein-1α/CCL3. When HGF was removed by immunoprecipitation, migration of THP-1 in RA SF was suppressed. In SKG mice, savolitinib significantly suppressed ankle bone destruction on µCT, with an associated reduction in the number of tartrate-resistant acid phosphatase-positive osteoclasts. HGF produced by inflammation in synovium of RA patients activates monocyte migration to synovium and promotes bone destruction via a chemotactic effect and enhanced chemokine production.

Atherosclerosis impacts the link between hepatocyte growth factor and cognition

Background. There is a controversy about the association between Hepatocyte growth factor (HGF) and cognition. Increased serum level of HGF has been reported in patients with hypertension, peripheral arteriosclerosis and carotid atherosclerosis. Ankle-brachial index (ABI) is considered as a marker of atherosclerosis. we hypothesized that hypertension or atherosclerosis with hypertension could alter the relation between HGF serum level and cognitive function. Aim. To study HGF and cognitive function in hypertensives with and without atherosclerosis versus healthy controls. Methods. This case-control study included ninety elderly subjects attending outpatient primary care geriatric clinics. They were subdivided into 3 groups; Group A (30 normotensives with normal Ankle-brachial index (ABI) as controls, group B (30 hypertensives with normal ABI) and group C (30 hypertensives with abnormal ABI) as cases. Cognitive function was assessed by Rowland Universal Dementia Assessment Scale (RUDAS). Results. Group C had worse score in RUDAS than controls (P = 0.01). HGF was negatively correlated with ABI in group C (p = 0.007). HGF was positively associated with RUDAS score, in group A (p < 0.001), in group B; after further adjustment for systolic blood pressure (SBP) (p = 0.024) and in group C; after adjustment for ABI (p = 0.031) or ABI and SBP (p = 0.05). Conclusions. The potential beneficial link between HGF serum concentration and cognition was met in normotensive subjects with normal ABI. However, this link is halted in the presence of hypertension or atherosclerosis as assessed by ABI. Alternatively, the raised HGF serum level may be an epiphenomenon of atherosclerosis.

Function of hepatocyte growth factor in gastric cancer proliferation and invasion.

Cancer incidence has been increasing steadily and is the leading cause of mortality worldwide. Gastric cancer is still most common malignancy in Korea. Cancer initiation and progression are multistep processes involving various growth factors and their ligands. Among these growth factors, we have studied hepatocyte growth factor (HGF), which is associated with cell proliferation and invasion, leading to cancer and metastasis, especially in gastric cancer. We explored the intercellular communication between HGF and other surface membrane receptors in gastric cancer cell lines. Using complimentary deoxyribonucleic acid microarray technology, we found new genes associated with HGF in the stomach cancer cell lines, NUGC-3 and MKN-28, and identified their function within the HGF pathway. The HGF/N-methyl-N’-nitroso-guanidine human osteosarcoma transforming gene (c-MET) axis interacts with several molecules including E-cadherin, urokinase plasminogen activator, KiSS-1, Jun B, and lipocalin-2. This pathway may affect cell invasion and metastasis or cell apoptosis and is therefore associated with tumorigenesis and metastasis in gastric cancer.

Remodeling of Glycosaminoglycans During Differentiation of Adult Human Bone Mesenchymal Stromal Cells Toward Hepatocytes.

There is a critical need to generate functional hepatocytes to aid in liver repair and regeneration upon availability of a renewable, and potentially personalized, source of human hepatocytes (hHEP). Currently, the vast majority of primary hHEP are obtained from human tissue through cadavers. Recent advances in stem cell differentiation have opened up the possibility to obtain fully functional hepatocytes from embryonic or induced pluripotent stem cells, or adult stem cells. With respect to the latter, human bone marrow mesenchymal stromal cells (hBMSCs) can serve as a source of autogenetic and allogenic multipotent stem cells for liver repair and regeneration. A major aspect of hBMSC differentiation is the extracellular matrix (ECM) composition and, in particular, the role of glycosaminoglycans (GAGs) in the differentiation process. In this study, we examine the influence of four distinct culture conditions/protocols (T1-T4) on GAG composition and hepatic markers. α-Fetoprotein and hepatocyte nuclear factor-4α were expressed continually over 21 days of differentiation, as indicated by real time quantitative PCR analysis, while albumin (ALB) expression did not begin until day 21. Hepatocyte growth factor (HGF) appears to be more effective than activin A in promoting hepatic-like cells through the mesenchymal-epithelial transition, perhaps due to the former binding to the HGF receptor to form a unique complex that diversifies the biological functions of HGF. Of the four protocols tested, uniform hepatocyte-like morphological changes, ALB secretion, and glycogen storage were found to be highest with protocol T2, which involves both early- and late-stage combinations of growth factors. The total GAG profile of the hBMSC ECM is rich in heparan sulfate (HS) and hyaluronan, both of which fluctuate during differentiation. The GAG profile of primary hHEP showed an HS-rich ECM, and thus, it may be possible to guide hBMSC differentiation to more mature hepatocytes by controlling the GAG profile expressed by differentiating cells.

Targeting the hepatocyte growth factor/Met pathway in cancer.

Hepatocyte growth factor (HGF)-induced activation of its cell surface receptor, the Met tyrosine kinase, drives mitogenesis, motogenesis and morphogenesis in a wide spectrum of target cell types and embryologic, developmental and homeostatic contexts. Typical paracrine HGF/Met signaling is regulated by HGF activation at target cell surfaces, HGF binding-induced receptor activation, internalization and degradation. Despite these controls, HGF/Met signaling contributes to oncogenesis, tumor angiogenesis and invasiveness, and tumor metastasis in many types of cancer, leading to the rapid growth of pathway-targeted anticancer drug development programs. We review here HGF and Met structure and function, basic properties of HGF/Met pathway antagonists now in clinical development, and recent clinical trial results. Presently, the main challenges facing the effective use of HGF/Met-targeted antagonists for cancer treatment include optimal patient selection, diagnostic and pharmacodynamic biomarker development, and the identification and testing of effective therapy combinations. The wealth of basic information, analytical reagents and model systems available regarding normal and oncogenic HGF/Met signaling will continue to be invaluable in meeting these challenges and moving expeditiously toward more effective cancer treatment.

Abstract 290: A novel function of HGF in the activation of Chk1 phosphorylation in colon cancer cells

Abstract ATR/Chk1 pathway plays an essential role in modulation of DNA damage response (DDR) and homologous recombination (HR). Particularly, Chk1 phosphorylation is involved in cancer prognosis and therapeutic resistance. Although some receptor tyrosine kinases (RTKs) participate in the regulation of Chk1 phosphorylation, the effect of hepatocyte growth factor (HGF) on Chk1 phosphorylation is unknown. In the present study, Western blotting and subcellular fractionation were performed to identify the expression and the location of Chk1 phosphorylation in the presence of HGF. With small interfering RNA transfections, co-immunoprecipitations were performed to detect the interaction between TopBP1 and ATR complex. Our results demonstrated that HGF moderately activated Chk1 phosphorylation in colon cancer cells via upregulation of TopBP1, RAD51, and complex formation of TopBP1 and ATR. Furthermore, AKT activation promoted by HGF served as an important intermediator linking HGF/MET signaling and Chk1 phosphorylation. Depletion of AKT activity attenuated expression of basal line p-Chk1 and induced Chk1 activation due to HGF stimulation. Meantime, activation of AKT directly regulated expression of TopBP1 and RAD51. Suppression of AKT restored HGF-induced upregulation of TopBP1, RAD51, and enhancement of TopBP1/ATR complex as well. Our studies showed that HGF was implicated in regulating Chk1 phosphorylation and further demonstrated that AKT activity was responsible for HGF-induced Chk1 phosphorylation, which might potentially result in management of prognosis and therapeutic sensitivity in cancer therapy. Note: This abstract was not presented at the meeting. Citation Format: Na Song, Xiaofang Che, Xiujuan Qu, Lu Xu, Kezuo Hou, Yunpeng Liu. A novel function of HGF in the activation of Chk1 phosphorylation in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 290. doi:10.1158/1538-7445.AM2017-290

A novel function of hepatocyte growth factor in the activation of checkpoint kinase 1 phosphorylation in colon cancer cells

The ATR/checkpoint kinase 1 (Chk1) pathway plays an essential role in modulating the DNA damage response and homologous recombination. Particularly, Chk1 phosphorylation is related to cancer prognosis and therapeutic resistance. Some receptor tyrosine kinases participate in the regulation of Chk1 phosphorylation; however, the effect of hepatocyte growth factor (HGF) on Chk1 phosphorylation is unknown. In the present study, we demonstrated that HGF moderately activated Chk1 phosphorylation in colon cancer cells by upregulating TopBP1 and RAD51, and promoting TopBP1–ATR complex formation. Furthermore, AKT activity, which was promoted by HGF, served as an important mediator linking HGF/MET signaling and Chk1 phosphorylation. Depleting AKT activity attenuated basal expression of p-Chk1 and HGF-induced Chk1 activation. Moreover, AKT activity directly regulated TopBP1 and RAD51 expression. AKT inhibition suppressed HGF-induced upregulation of TopBP1 and RAD51, and enhanced TopBP1/ATR complex formation. Our results show that HGF was involved in regulating Chk1 phosphorylation, and further demonstrate that AKT activity was responsible for this HGF-induced Chk1 phosphorylation. These findings might potentially result in management of prognosis and therapeutic sensitivity in cancer therapy.

Effect of STAT3 inhibitor in chronic myeloid leukemia associated signaling pathway: a mathematical modeling, simulation and systems biology study.

Chronic myeloid leukemia (CML) is a hematopoietic stem-cell disorder which proliferates due to abnormal growth of basophil cells. Several proangiogenic molecules have been reported to be associated in CML progression, including the hepatocyte growth factor (HGF). However, detail mechanism about the cellular distribution and function of HGF in CML is yet to be revealed. The proliferation of hematopoietic cells are regulated by some of the growth factors like interleukin 3 (IL-3), IL-6, erythropoietin, thrombopoietin, etc. In this study IL-6 pathways have been taken into consideration which induces JAK/STAT and MAPK pathways to decipher the CML progression stages. An attempt has been made to model these pathways with the help of ordinary differential equations (ODEs) and estimating unknown parameters through fminsearch optimization algorithm. Some of the specific component like STAT3, of the pathway has been analyzed in detail and their role in CML progression has been elucidated. The roles of STAT3 inhibitors into the treatment of CML have been thoroughly studied and optimum concentration of the inhibitors have been predicted.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-015-0357-7) contains supplementary material, which is available to authorized users.

Effects of SNPs and alternative splicing within HGF gene on its expression patterns in Qinchuan cattle

BackgroundIdentification of genetic variants, including SNPs (Single Nucleotide Polymorphisms), CNVs (Copy Number Variations) and alternative splicing, within functional genes has received increasing attention in animal science research. HGF (Hepatocyte Growth Factor) is a very important growth factor that works as a mitogen or a morphogen during tissue growth, development and regeneration. However, to date, the functions of genetic variants within the bovine HGF gene, particularly their effects on mRNA expression, have not been determined well.ResultsThe present study aimed to perform association analysis between genetic variants and mRNA expression for the bovine HGF gene in Qinchuan cattle using various strategies, including PCR-RFLP (Restriction Fragment Length Polymorphism), qPCR (Quantitative Real-time quantitative PCR), TA cloning, DNA sequencing and bioinformatics analysis. A total of five SNPs were identified and only SV1 locus significantly affected HGF mRNA expression in fetal skeletal muscle (P < 0.05). Heterozygous genotype individuals showed significantly higher HGF expression (P < 0.05), which was significantly greater in the “CTCCAGGGTT” combined genotype than that in the “CCCCGGGGTT” combined genotype (P < 0.05). In addition, two alternative splicing variations, HGF-W and HGF-M, were identified, which resulted from alternative 3′ splice sites of exon 5, and HGF-W showed higher mRNA levels than HGF-M in all tissues.ConclusionIn summary, genetic variations within the HGF gene affected mRNA expression. These findings provide new insight into the molecular characteristics and functions of bovine HGF.

Integrated analysis of gene expression and DNA methylation changes induced by hepatocyte growth factor in human hepatocytes.

Hepatocellular carcinoma (HCC) is the one of most common malignant tumors. The tumor microenvironment has a role in not only supporting growth and survival of tumor cells, but also triggering tumor recurrence and metastasis. Hepatocyte growth factor (HGF), one of the important growth factors in the tumor microenvironment, has an important role in angiogenesis, tumorigenesis and regeneration. However, the exact mechanism by which HGF regulates HCC initiation and development via epigenetic reprogramming has remained elusive. The present study focused on the epigenetic modification and target tumor-suppressive genes of HGF treatment in HCC. Expression profiling and DNA methylation array were performed to investigate the function of HGF and examine global genomic DNA methylation changes, respectively. Integrated analysis of gene expression and DNA methylation revealed potential tumor suppressor genes (TSGs) in HCC. The present study showed the multiple functions of HGF in tumorous and nontumorous pathways and global genomic DNA methylation changes. HGF treatment upregulated the expression of DNA methyltransferase 1 (DNMT1). Overexpression of DNMT1 in HCC patients correlated with the malignant potential and poor prognosis of HCC. Furthermore, integration analysis of gene expression and DNA methylation changes revealed novel potential tumor suppressor genes TSGs including MYOCD, PANX2 and LHX9. The present study has provided mechanistic insight into epigenetic repression of TSGs through HGF-induced DNA hypermethylation.

Identification of Basophils as a Major Source of Hepatocyte Growth Factor in Chronic Myeloid Leukemia: A Novel Mechanism of BCR-ABL1-Independent Disease Progression

Chronic myeloid leukemia (CML) is a hematopoietic neoplasm characterized by the Philadelphia chromosome and the related BCR-ABL1 oncoprotein. Acceleration of CML is usually accompanied by basophilia. Several proangiogenic molecules have been implicated in disease acceleration, including the hepatocyte growth factor (HGF). However, little is known so far about the cellular distribution and function of HGF in CML. We here report that HGF is expressed abundantly in purified CML basophils and in the basophil-committed CML line KU812, whereas all other cell types examined expressed only trace amounts of HGF or no HGF. Interleukin 3, a major regulator of human basophils, was found to promote HGF expression in CML basophils. By contrast, BCR-ABL1 failed to induce HGF synthesis in CML cells, and imatinib failed to inhibit expression of HGF in these cells. Recombinant HGF as well as basophil-derived HGF induced endothelial cell migration in a scratch wound assay, and these effects of HGF were reverted by an anti-HGF antibody as well as by pharmacologic c-Met inhibitors. In addition, anti-HGF and c-Met inhibitors were found to suppress the spontaneous growth of KU812 cells, suggesting autocrine growth regulation. Together, HGF is a BCR-ABL1-independent angiogenic and autocrine growth regulator in CML. Basophils are a unique source of HGF in these patients and may play a more active role in disease-associated angiogenesis and disease progression than has so far been assumed. Our data also suggest that HGF and c-Met are potential therapeutic targets in CML.

Hepatocyte growth factor overexpression in the nervous system enhances learning and memory performance in mice

Hepatocyte growth factor (HGF) and its receptor, c-Met, play pivotal roles in the nervous system during development and in disease states. However, the physiological roles of HGF in the adult brain are not well understood. In the present study, to assess its role in learning and memory function, we used transgenic mice that overexpress HGF in a neuron-specific manner (HGF-Tg) to deliver HGF into the brain without injury. HGF-Tg mice displayed increased alternation rates in the Y-maze test compared with age-matched wild-type (WT) controls. In the Morris water maze (MWM) test, HGF-Tg mice took less time to find the platform on the first day, whereas the latency to escape to the hidden platform was decreased over training days compared with WT mice. A transfer test revealed that the incidence of arrival at the exact location of the platform was higher for HGF-Tg mice compared with WT mice. These results demonstrate that overexpression of HGF leads to an enhancement of both short- and long-term memory. Western blot analyses revealed that the levels of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, but not NR1, were increased in the hippocampus of HGF-Tg mice compared with WT controls, suggesting that an upregulation of NR2A and NR2B could represent one mechanism by which HGF enhances learning and memory performance. These results demonstrate that modulation of learning and memory performance is an important physiological function of HGF that contributes to normal CNS plasticity, and we propose HGF as a novel regulator of higher brain functions.

Fibroblasts in omentum activated by tumor cells promote ovarian cancer growth, adhesion and invasiveness

Omentum metastasis is a common occurrence in epithelial ovarian cancer (EOC), which is often accompanied by ascites that facilitates the spread of EOC cells. A subpopulation of fibroblasts-the cancer-associated fibroblasts (CAFs) are important promoters of tumor progression. We have shown previously that CAFs exist not only in omentum with EOC metastasis but also in omentum without metastasis. In the present study, using primary human fibroblasts isolated from normal omentum (NFs) and omentum with ovarian cancer metastasis (CAFs), we established in vitro coculture models and a 3D culture model mimicking human omentum structure for investigation of interactions between fibroblasts and cancer cells. We demonstrate that EOC cells activate NFs and promote their proliferation via transforming growth factor-β1 (TGF-β1) signaling, and the activated fibroblasts contribute to the invasion and adhesion of EOC cells. Moreover, EOC cells and NFs coculture led to overexpression of hepatocyte growth factor (HGF) and matrix metalloproteinase-2 (MMP-2) and adhesion and invasion of EOC cells could be partially suppressed by blocking the function of HGF or MMP-2. Additionally, mouse peritoneal dissemination models of EOC confirmed the activation of fibroblasts by cancer cells and the tumor growth- and metastasis-promoting effects of activated fibroblasts in vivo. Our findings indicate that activated fibroblasts in omentum form a congenial environment to promote EOC cells implantation. It is an intriguing concept that targeting the activation of omentum fibroblast through the inhibition of TGF-β1 signaling can be used as a new therapeutic strategy against ovarian cancer omentum metastases, which needs further study.

Hepatocyte growth factor promoting the proliferation of human eccrine sweat gland epithelial cells is relative to AKT signal channel and β-catenin

Hepatocyte growth factor (HGF) is a multi-effective molecule, playing important roles in organ growth, tumorigenesis and trauma healing. This experiment aims at studying the promoting function of HGF on the proliferation of human eccrine sweat gland epithelial cells (hESGc) and its relative signal channels. After HGF at different concentrations were added into cells, MTT was adopted to detect the cell proliferations, Annexin-V/PI the cell apoptosis, and Westernblot the expressions of p-AKT, AKT, p-ERK, p-GSK3β, p-IKBα, and β-catenin in hESGc. After adding siRNA c-Met to block HGF or LY294002 to inhibit p-AKT, we used MTT to detect the proliferation of hESGc and Westernblot to detect the expression of β-catenin. As a result, 20-40 ng/mL HGF could promote the proliferation of hESGc and inhibit its apoptosis. HGF could promote the expressions of p-AKT1/2/3, p-ERK, p-GSK3β, p-IKBα, and β-catenin. The additions of siRNA c-Met to block HGF or LY294002 to inhibit p-AKT could downregulate β-catenin and inhibit the proliferation promotion caused by HGF. Consequently, we concluded HGF can promote the proliferation of human eccrine sweat gland epithelial cells, which is relative to AKT signal channel and β-catenin.

The impact of compositional topography of amniotic membrane scaffold on tissue morphogenesis of salivary gland

Amniotic membrane (AM) has been widely used in the reconstruction of oral epithelial defects. However, whether it is also effective in facilitating tissue formation of salivary gland, an appendix of oral epithelia, has never been explored. To investigate the effects and the underlying mechanism of AM on salivary gland morphogenesis, murine fetal submandibular gland (SMG) explants were cultured on different preparations of AM scaffolds. It was found that, on AM stromal scaffold, SMG demonstrated well-developed branching morphogenesis. Nonetheless, on AM epithelial scaffold, SMG epithelial cell converted to a spindle-shape, lost intercellular connection, changed cytoskeletal organization, and exhibited scattering behaviors. Meanwhile, the integrity of SMG basement membrane was dismantled as well. However, when acellular AM epithelial scaffold was used, cultured SMG demonstrated organized morphology, indicating that AM epithelial component provided specific surface features for SMG morphogenesis. To further investigate AM scaffold morphogenetic effect, it was found hepatocyte growth factor (HGF), an epithelial scattering factor, was expressed abundantly in cultivated AM epithelia. After blocking HGF function of AM, cultured SMG regained branching activity, reorganized cell adhesion and subcellular organization, and reproduced basement membranes. Therefore, AM-derived bioactive factor profoundly influences cell behaviors and structure formation of SMG. Together, this study showed that compositional topography of AM scaffold is important in affecting SMG morphogenesis. By understanding the effects of AM scaffold on SMG morphogenesis, it provides important information for rationally designing and fabricating AM scaffold for salivary gland regeneration.

Demonstration of the hepatocyte growth factor signaling pathway in the in vitro neuritogenic activity of chondroitin sulfate from ray fish cartilage

BackgroundChondroitin sulfate (CS) is a ubiquitous component of the cell surface and extracellular matrix and its sugar backbone consists of repeating disaccharide units: D-glucuronic acid (GlcUA)β1-3N-acetyl-D-galactosamine (GalNAc). Although CS participates in diverse biological processes such as growth factor signaling and the nervous system's development, the mechanism underlying the functions is not well understood. MethodsCS was isolated from ray fish cartilage, an industrial waste, and its structure and neurite outgrowth-promoting (NOP) activity were analyzed to investigate a potential application to nerve regeneration. ResultsThe major disaccharide unit in the CS preparation was GlcUA-GalNAc(6-O-sulfate) (61.9%). Minor proportions of GlcUA-GalNAc(4-O-sulfate) (27.0%), GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate) (8.5%), and GlcUA-GalNAc (2.7%) were also detected. The preparation showed NOP activity in vitro, and this activity was suppressed by antibodies against hepatocyte growth factor (HGF) and its receptor c-Met, suggesting the involvement of the HGF signaling pathway in the expression of the in vitro NOP activity of the CS preparation. The specific binding of HGF to the CS preparation was also demonstrated by surface plasmon resonance spectroscopy. Conclusions and general significanceThe NOP activity of CS from ray cartilage was demonstrated to be expressed through the HGF signaling pathway, suggesting that ray cartilage CS may be useful for studying the cooperative function of CS and HGF.

Stromal Hyaluronan Interaction with Epithelial CD44 Variants Promotes Prostate Cancer Invasiveness by Augmenting Expression and Function of Hepatocyte Growth Factor and Androgen Receptor

The main aim of our study is to determine the significance of the stromal microenvironment in the malignant behavior of prostate cancer. The stroma-derived growth factors/cytokines and hyaluronan act in autocrine/paracrine ways with their receptors, including receptor-tyrosine kinases and CD44 variants (CD44v), to potentiate and support tumor epithelial cell survival. Overexpression of hyaluronan, CD44v9 variants, and stroma-derived growth factors/cytokines are specific features in many cancers, including prostate cancer. Androgen/androgen receptor interaction has a critical role in regulating prostate cancer growth. Our previous study showed that 1) that increased synthesis of hyaluronan in normal epithelial cells promotes expression of CD44 variants; 2) hyaluronan interaction with CD44v6-v9 promotes activation of receptor-tyrosine kinase, which stimulates phosphatidylinositol 3-kinase-induced cell survival pathways; and 3) CD44v6/short hairpin RNA reduces colon tumor growth in vivo (Misra, S., Hascall, V. C., De Giovanni, C., Markwald, R. R., and Ghatak, S. (2009) J. Biol. Chem. 284, 12432–12446). Our results now show that hepatocyte growth factor synthesized by myofibroblasts associated with prostate cancer cells induces activation of HGF-receptor/cMet and stimulates hyaluronan/CD44v9 signaling. This, in turn, stabilizes the androgen receptor functions in prostate cancer cells. The stroma-derived HGF induces a lipid raft-associated signaling complex that contains CD44v9, cMet/phosphatidylinositol 3-kinase, HSP90 and androgen receptor. CD44v9/short hairpin RNA reverses the assembly of these components in the complex and inhibits androgen receptor function. Our results provide new insight into the hyaluronan/CD44v9-regulated androgen receptor function and the consequent malignant activities in prostate cancer cells. The present study describes a physiologically relevant in vitro model for studying the molecular mechanisms by which stroma-derived HGF and hyaluronan influence androgen receptor and CD44 functions in the secretory epithelia during prostate carcinogenesis.

Diverse Roles of Hepatocyte Growth Factor (HGF) in Normal Karyotype Acute Myeloid Leukemia (AML).

Abstract 3107Poster Board III-44Prior studies have demonstrated that hepatocyte growth factor (HGF) regulates proliferation and differentiation of normal hematopoietic progenitors. HGF activity occurs primarily via interactions with the c-met receptor, a tyrosine kinase receptor found on epithelial and some cancer cells. In solid tumors, HGF/c-met interactions mediate increased neoplastic invasion, metastases, and angiogenesis. However, in vitro, HGF has also been shown to mediate anti-tumor effects in leukemia cell line models. To better elucidate the role of HGF in acute leukemogenesis, we evaluated HGF and c-met gene expression in 91 normal karyotype acute myeloid leukemia (NK-AML) patient samples previously characterized for marrow angiogenesis (CD31+ microvessels), FLT-3/NPM-1 gene mutation, and pro-angiogenic factors and receptors (specifically vascular endothelial growth factors (VEGF-A and C) and their receptors). Median patient age was 66 years (range 21-87) with 49 women and 42 men. AML disease FAB subtypes M2 (37%) and M1 (36%) subtypes were most common. Median presenting white blood cell count (WBC) was 32,000/μL (range 0.43-555,000/μL) with marrow blasts of 70.6% (range 15-95.4%). Fourteen percent presented with extramedullary disease. Median OS was 9.4 months (95% CI 6.7 to 11.5 months), with median EFS of 8 months (95% CI 5.7 to 11.5 months) for all patients. Seventy-nine patients received cytarabine and anthracycline-based induction chemotherapy with 58% (n=46) achieving complete remission (CR). Marrow aspirate samples were evaluated by quantitative real-time polymerase chain reaction with levels expressed relative to normal bone marrow controls (set =1). We found that HGF gene expression was upregulated in most primary NK-AML patient samples, with 88% expressing higher HGF than normal bone marrow. Median HGF expression in AML samples was 7.73 fold higher than normal controls. Multivariate analysis including age, complete remission, marrow blasts, extramedullary disease, and expression of other angiogenic factors and receptors as covariables, showed high HGF expression to be significantly associated with both longer overall and event-free survival. Surprisingly, HGF gene expression was found to be negatively correlated with microvessel density and NPM-1 mutation and positively correlated with the VEGF receptor neuropilin-1 (NRP-1) which has been reported to function as co-mediator of HGF activity. No association between HGF and FLT-3 ITD mutation was noted. The majority of AML samples did not express the HGF receptor, c-met, suggesting that HGF function in AML occurs primarily via paracrine interactions with surrounding vascular and stromal cells and/or HGF/NRP-1 autocrine pathways. Further analysis confirmed no significant correlation between HGF and c-met gene expression in AML samples but did demonstrate a subset of NPM-1+ HGF+ AML samples (n=7) expressing high levels of both HGF and c-met (p=0.0005, r=0.96). To confirm whether HGF/c-met autocrine interactions contributed to leukemogenesis in these cells, we treated immunodeficient mice engrafted with an HGF+ c-met+ human AML cell line (HEL) with vehicle vs. a c-met tyrosine kinase inhibitor, and noted growth inhibitory effects following c-met blockade. ConclusionsHGF gene expression was an independent predictive factor for improved clinical outcome and was associated with NRP-1 expression, lower microvessel density, and NPM-1 negative status in normal karyotype AML patients. A subset of AML samples was identified with high concordant HGF and c-met expression consistent with autocrine pathways. Inhibition of HGF/c-met interactions in a preclinical AML model exerted anti-tumor effects. Additional studies of the diverse roles of HGF in myeloid leukemogenesis are warranted. DisclosuresNo relevant conflicts of interest to declare.