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Supplementary Table 2 from Glutathione Peroxidase 3, Deleted or Methylated in Prostate Cancer, Suppresses Prostate Cancer Growth and Metastasis

Glutathione peroxidase 3 is a selenium-dependent enzyme playing a critical role in detoxifying reactive oxidative species and maintaining the genetic integrity of mammalian cells. In this report, we found that the expression of glutathione peroxidase 3 (GPx3) was widely inactivated in prostate cancers. Complete inactivation of GPx3 correlates with a poor clinical outcome. Deletions (hemizygous and homozygous) of GPx3 gene are frequent in prostate cancer samples, occurring in 39% of the samples studied. The rate of methylation of the GPx3 exon 1 region in prostate cancer samples reaches 90%. Overexpression of GPx3 in prostate cancer cell lines induced the suppression of colony formation and anchorage-independent growth of PC3, LNCaP, and Du145 cells. PC3 cells overexpressing GPx3 reduced invasiveness in Matrigel transmigration analysis by an average of 2.7-fold. Xenografted PC3 cells expressing GPx3 showed reduction in tumor volume by 4.8-fold, elimination of metastasis (0/16 versus 7/16), and reduction of animal death (3/16 versus 16/16). The tumor suppressor activity of GPx3 seems to relate to its ability to suppress the expression of c-met. The present findings suggest that GPx3 is a novel tumor suppressor gene. [Cancer Res 2007;67(17):8043–50]

Supplementary Table 1 from Glutathione Peroxidase 3, Deleted or Methylated in Prostate Cancer, Suppresses Prostate Cancer Growth and Metastasis

Supplementary Figure 1 from Glutathione Peroxidase 3, Deleted or Methylated in Prostate Cancer, Suppresses Prostate Cancer Growth and Metastasis

Purpose Status epilepticus (SE) is a commonly encountered neurologic condition associated with high mortality rates. Cyclic seizures (CS) are a common form of SE, but its prognostic significance has not been well established. In this retrospective study, the mortality of cyclic versus noncyclic forms (NCSs) of SE are compared. Methods A total of 271 patients were identified as having seizures or SE on EEG reports, of which 65 patients were confirmed as having SE. Based on EEG characteristics, the patients were then classified as cyclic or noncyclic patterns. Cyclic seizures were defined as recurrent seizures occurring at nearly regular and uniform intervals. Noncyclic form included all other patterns of SE. Pertinent clinical data were collected and reviewed for each case. Results Of the 65 patients with SE, 25 patients had CS and 40 patients had NCS. Patients with CS showed a lower rate of in-hospital mortality although not statistically significant (P = 0.19). When looking at patients younger than 75 years, the CS group had significantly lower in-hospital mortality rate (P = 0.007). Conclusions The findings of this study suggest that CS may have a more favorable outcome compared with NCS in patients younger than 75 years. This study is also the first to report the rate of CS among all cases of confirmed SE (38%). Future studies with a larger sample size are needed to further evaluate the difference in outcome between CS and NCS.

Background & Aims Nonalcoholic fatty liver disease is a frequent cause of hepatic dysfunction and is now a global epidemic. This ailment can progress to an advanced form called nonalcoholic steatohepatitis (NASH) and end-stage liver disease. Currently, the molecular basis of NASH pathogenesis is poorly understood, and no effective therapies exist to treat NASH. These shortcomings are due to the paucity of experimental NASH models directly relevant to humans. Methods We used chimeric mice with humanized liver to investigate nonalcoholic fatty liver disease in a relevant model. We carried out histologic, biochemical, and molecular approaches including RNA-Seq. For comparison, we used side-by-side human NASH samples. Results Herein, we describe a “humanized” model of NASH using transplantation of human hepatocytes into fumarylacetoacetate hydrolase-deficient mice. Once fed a high-fat diet, these mice develop NAFLD faithfully, recapitulating human NASH at the histologic, cellular, biochemical, and molecular levels. Our RNA-Seq analyses uncovered that a variety of important signaling pathways that govern liver homeostasis are profoundly deregulated in both humanized and human NASH livers. Notably, we made the novel discovery that hepatocyte growth factor (HGF) function is compromised in human and humanized NASH at several levels including a significant increase in the expression of the HGF antagonists known as NK1/NK2 and marked decrease in HGF activator. Based on these observations, we generated a potent, human-specific, and stable agonist of human MET that we have named META4 (Metaphor) and used it in the humanized NASH model to restore HGF function. Conclusions Our studies revealed that the humanized NASH model recapitulates human NASH and uncovered that HGF-MET function is impaired in this disease. We show that restoring HGF-MET function by META4 therapy ameliorates NASH and reinstates normal liver function in the humanized NASH model. Our results show that the HGF-MET signaling pathway is a dominant regulator of hepatic homeostasis., Graphical abstract

Objective To characterize patients with coronavirus disease 2019 (COVID-19) who presented primarily with neurologic symptoms without typical COVID-19 symptoms of fever, cough, and dyspnea. Methods We retrospectively identified COVID-19-positive patients 18 years and older that had neurology symptoms on presentation requiring neurology consultation between March 14, 2020 and May 18, 2020. The patients were then classified into those with typical COVID-19 symptoms and those without. Demographic, clinical symptoms, laboratory result, and clinical outcomes were collected. Results Out of 282 patients who had neurology consult during this period, we identified 56 (mean age 69.2 years, 57% women) who tested COVID-19-positive and had neurologic symptoms on initial presentation. Of these, 23 patients (mean age 65.2 years, 52% women) had no typical COVID-19 symptoms while 33 did (mean age 72.2 years, 60% woman). In both groups, impaired consciousness was the most common initial neurologic symptom, followed by stroke, unsteady gait, headache, seizure, syncopal event, acute vision changes, and intracranial hemorrhage. Out of the 23 patients without typical COVID-19 symptoms on presentation, 10 went on to develop typical symptoms with 8 needing supplemental oxygen and one requiring mechanical ventilation. Conclusion Patients who have COVID-19 can present with serious neurologic symptoms such as impaired consciousness and stroke even without typical COVID-19 symptoms. Those without typical COVID-19 symptoms can later develop typical symptoms severe enough to need respiratory support.

Nitrous oxide, often used as an anesthetic agent, is also increasingly a drug of abuse due to its euphoric and anxiolytic effects. Frequent exposure to nitrous oxide can lead to neurologic complications, including B12 deficiency and resultant subacute myeloneuropathy, as well as direct neurotoxicity. A clinical presentation of acute sensorimotor polyneuropathy mimicking Guillain-Barré syndrome after chronic nitrous oxide abuse has been reported only rarely. Here we present a 17-year-old previously healthy girl presented with 10 days of progressive ascending sensory loss and weakness in the legs. She admitted to heavy nitrous oxide abuse over a period of a year or more. Laboratory evaluation was significant for normal vitamin B12 level with elevated homocysteine. A magnetic resonance imaging (MRI) of her spine showed abnormal signal involving the bilateral dorsal columns. Nerve conduction studies were suggestive of severe axonal sensorimotor polyneuropathy. This patient demonstrates concurrent multifactorial neurologic injury as a result of nitrous oxide abuse. She had a functional vitamin B12 deficiency as indicated by the elevated homocysteine, leading to a subacute combined degeneration that was evident on the MRI. In addition, she had evidence of direct neurotoxicity leading to axonal injury and sensorimotor polyneuropathy reminiscent of Guillain-Barré syndrome. This clinical picture is a serious but seldom reported possible complication if nitrous oxide abuse and should be considered in patients presenting with a clinical picture suspicious for Guillain-Barré syndrome or its variants.

Purpose Generalized periodic discharges (GPDs) are frequently identified in the EEGs of hospitalized patients but their prognostic significance remains unclear. We retrospectively reviewed clinical data in patients with GPDs to elucidate factors associated with in-hospital mortality. Method We reviewed data from inpatients at three different hospitals affiliated with our institution in whom GPDs were reported on routine EEGs by fellowship-trained electroencephalographers during the years 2010–2012. Cox regression was used to determine statistical association between in-hospital death and demographics, medical comorbidities, neurological and neuroimaging abnormalities and antiepileptic drug use. Results We identified 113 patients with GPDs. The mean age was 70.4 years and 70 (61.9%) were women. There were 60 inpatient deaths (53.1%). The variables significantly associated with in-hospital mortality were dementia, poor mental status at the time of the EEG, chronic focal abnormalities on neuroimaging, cardiac arrest and chronic obstructive pulmonary disease (COPD). Conclusion Dementia, poor mental status during EEG, chronic focal abnormalities on neuroimaging, cardiac arrest and COPD are independently associated with increased in-hospital mortality in patients with GPDs ( P

Background: Few studies in the literature discuss operative positioning for lumbar surgery precipitating acute cauda equina syndromes (CES). Case Description: A 56-year-old male with a large L2-3-disc herniation was placed prone on a Jackson table. He immediately lost all motor and sensory evoked potentials. Signals returned to the baseline when surgery was aborted, and he was returned to the supine position. However, potentials were again lost when he was repositioned prone, following which the surgeons proceeded with surgical decompression with a good outcome. Conclusion: This case highlights the risk for patients with large acute lumbar disc herniation/stenosis and CES undergoing prone positioning for lumbar decompression. Here, despite the secondary loss of both sensory and motor evoked potentials, the patient successfully underwent lumbar decompressive surgery/discectomy performed on a Jackson table, resulting in full postoperative neurological recovery.

Met is the transmembrane tyrosine kinase cell surface receptor for hepatocyte growth factor (HGF) and is structurally related to the insulin receptor (INSR) tyrosine kinase. Here we report that the HGF-Met axis regulates metabolism by stimulating hepatic glucose uptake and suppressing hepatic glucose output. We show that Met is essential for an optimal hepatic insulin response by directly engaging INSR to form a Met-INSR hybrid complex, which culminates in a robust signal output. We also found that the HGF-Met system restores insulin responsiveness in a mouse model of insulin refractoriness. These results provide new insights into the molecular basis of hepatic insulin resistance and suggest that HGF may have therapeutic potential for type 2 diabetes in the clinical setting.

Introduction With the rising use of continuous EEG monitoring, the ability to detect seizures rapidly and treat is becoming a standard of care. The issue for many institutions is the availability of an EEG reader to review EEG studies continuously. Persyst is a seizure detection software that allows health care professionals not trained in EEG to identify seizures rapidly at the bedside. We aim to determine if different electrographic patterns of status epilepticus (SE) affects detection rate by Persyst. Methods Continuous EEG monitoring reports at New York Presbyterian Queens Hospital from January 1st, 2015 to December 31st, 2016 with the phrase “status epilepticus” were selected. EEG patterns were categorized by board certified epileptologists as (1) SE associated with repetitive focal seizures (SERFS) or (2) SE associated with the Ictal-Interictal continuum (SEIIC). SERFS were defined as focal origin with a clear ictal onset and offset and a definable inter-seizure interval. SEIIC were defined as having association with characteristic features in the ictal-interictal continuum such as periodic discharges (generalized, lateralized, bilateral independent or stimulus-induced) and/or rhythmic delta activity. The archived raw EEG for each case was analyzed using Persyst Software. If at least one seizure was detected, it was classified as positive. If there were zero seizure detections, then it was classified as negative. Cases which had insufficient raw EEG data available were excluded from this study. Statistical analysis was performed using Chi-Square test. Results A total of 11 patients were identified with status epilepticus. Of the 11 patients, 5 were classified as SERFS and 6 were classified as SEIIC. Following analysis using Persyst, 5/5 patients with SERFS and 2/6 patients with SEIIC had positive seizure detections. Persyst was significantly more sensitive in detecting seizures in SERFS than in SIIC (p = 0.02). Conclusion This study showed that Persyst is highly sensitive in detecting SERFS but ineffective in SEIIC. These findings are consistent with prior study on this topic. This study, although small in sample size, further adds to the literature showing that the electrographic pattern of status epilepticus may influence the sensitivity of seizure detection using automated seizure detection software such as Persyst. Future studies with a larger sample size would be helpful to further clarify this hypothesis.

NF kappaB transcription factor regulates gene expression in response to extracellular stimuli such as TNF alpha. The genes regulated by NF kappaB encode for proteins which control cell growth and survival. Met is the tyrosine kinase receptor for hepatocyte growth factor, and it too promotes cell mitogenesis and survival. Previously, we showed that Met gene expression is regulated by TNF alpha. In this report, we identify and characterize a TNF alpha response element in the Met promoter. This element contains tandem C/EBP sites adjacent to an NF kappaB site. Binding of the NF kappaB p65 subunit and C/EBP beta to this element is induced by TNF alpha. To examine the interplay of NF kappaB and Met in vivo, we determined that Met mRNA and protein levels are reduced in the livers of p65-/- mice as compared to controls. In p65-/- mouse embryonic fibroblasts (MEFs), Met induction by TNF alpha is abrogated while Met's basal gene expression is reduced by half as compared to controls. When overexpressed in p65-/- MEFs, Met confers resistance to TNF-alpha-mediated cell death. Conversely, expression of dominant negative Met in wild-type MEFs renders them sensitive to cell death induced by TNF alpha. A similar response following TNF alpha challenge was observed in hepatocytic cells treated with siRNA to knockdown endogenous Met. Together, these results indicate that the Met gene is a direct target of NF kappaB and that Met participates in NF kappaB-mediated cell survival.

Hepatocytes in fatty livers are hypersensitive to apoptosis and undergo escalated apoptotic activity via death receptor-mediated pathways, particularly that of Fas-FasL, causing hepatic injury that can eventually proceed to cirrhosis and end-stage liver disease. Here we report that the hepatocyte growth factor receptor, Met, plays an important part in preventing Fas-mediated apoptosis of hepatocytes by sequestering Fas. We also show that Fas antagonism by Met is abrogated in human fatty liver disease (FLD). Through structure-function studies, we found that a YLGA amino-acid motif located near the extracellular N terminus of the Met alpha-subunit is necessary and sufficient to specifically bind the extracellular portion of Fas and to act as a potent FasL antagonist and inhibitor of Fas trimerization. Using mouse models of FLD, we show that synthetic YLGA peptide tempers hepatocyte apoptosis and liver damage and therefore has therapeutic potential.

Signaling initiated by Class Ia phosphatidylinositol-3-kinases (PI3Ks) is essential for cell proliferation and survival. We discovered a novel protein we call PI3K interacting protein 1 (PIK3IP1) that shares homology with the p85 regulatory PI3K subunit. Using a variety of in vitro and cell based assays, we demonstrate that PIK3IP1 directly binds to the p110 catalytic subunit and down modulates PI3K activity. Our studies suggest that PIK3IP1 is a new type of PI3K regulator.

Hypoxia/reoxygenation causes cellular injury and death associated with a number of pathophysiological conditions, including myocardial ischemia/reperfusion injury and stroke. The cell death pathways induced by hypoxia/reoxygenation and their underlying regulatory mechanisms remain poorly understood. Recent studies have shown that hypoxia/reoxygenation can induce Bax translocation and cytochrome c release. Using murine lung endothelial cells as a model, we found that the induction of apoptosis by hypoxia/reoxygenation involved the activation of both Bax-dependent and death receptor-mediated pathways. We demonstrated the activation of the death-inducing signal complex and Bid pathway after hypoxia/reoxygenation. Hepatocyte growth factor markedly inhibited hypoxia/reoxygenation-induced endothelial cell apoptosis. The cytoprotection afforded by hepatocyte growth factor was mediated in part by the stimulation of FLICE-like inhibiting protein expression, the attenuation of death-inducing signal complex formation, and the inhibition of Bid and Bax activation. Hepatocyte growth factor also prevented cell injury and death by increasing the expression of the antiapoptotic Bcl-XL protein. The inhibition of Bid/Bax-induced cell death by hepatocyte growth factor primarily involved p38 MAPK and in part Akt-dependent pathways but not ERK1/ERK2.

The rate of complications during thyroid surgery has decreased because of better instrumentation, illumination, and surgical expertise. Despite these improvements, certain patients, those requiring reoperation, those with invasive cancers or with numerous nodal metastasis or recurrent tumors, and those with large substernal goiters have a low but appreciable risk for complications. When planning a thyroid operation, one should perform the operation that corrects these problems and decreases the risk for complications. Localization of at least one parathyroid gland is essential. No surgical procedure can be done, however, without a risk of complications. To decrease these possible risks the surgeon should understand the embryologic development of the thyroid and parathyroid glands, the anatomical position of key structures, and use meticulous operative technique. Experience in performing thyroid operations is essential for the best outcome with the fewest complications.

Death receptors such as Fas are present in a variety of organs including liver and play an important role in homeostasis. What prevents these harmful receptors from forming homooligomers, clustering, and initiating the apoptotic pathway is not known. Here, we report the discovery of a cell survival mechanism by which Met, a growth factor receptor tyrosine kinase, directly binds to and sequesters the death receptor Fas in hepatocytes. This interaction prevents Fas self-aggregation and Fas ligand binding, thus inhibiting Fas activation and apoptosis. Our results describe a direct link between growth factor tyrosine kinase receptors and death receptors to establish a novel paradigm in growth regulation.

Met, the transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF), is known to function as a potent antiapoptotic mediator in normal and neoplastic cells. Herein we report that the intracellular cytoplasmic tail of Met has evolved to harbor a tandem pair of caspase-3 cleavage sites, which bait, trap, and disable the active site of caspase-3, thereby blocking the execution of apoptosis. We call this caspase-3 cleavage motif the Death Defying Domain (DDD). This site consists of the following sequence: DNAD-DEVD-T (where the hyphens denote caspase cleavage sites). Through functional and mechanistic studies, we show that upon DDD cleavage by caspase-3 the resulting DEVD-T peptide acts as a competitive inhibitor and entraps the active site of caspase-3 akin to DEVD-CHO, which is a potent, synthetic inhibitor of caspase-3 activity. By gain- and loss-of-function studies using restoration of DDD expression in DDD-deficient hepatocytic cells, we found that both caspase-3 sites in DDD are necessary for inhibition of caspase-3 and promotion of cell survival. Employing mutagenesis studies, we show that DDD could operate independently of Met's enzymatic activity as determined by using kinase-dead human Met mutant constructs. Studies of both human liver cancer tissues and cell lines uncovered that DDD cleavage and entrapment of caspase-3 by DDD occur in vivo, further proving that this site has physiological and pathophysiological relevance. Conclusion: Met can directly inhibit caspase-3 by way of a novel mechanism and promote hepatocyte survival. The results presented here will further our understanding of the mechanisms that control not only normal tissue homeostasis but also abnormal tissue growth such as cancer and degenerative diseases in which apoptotic caspases are at play. (Hepatology 2014;59:2010–2021)

Hepatocyte growth factor (HGF) is a pleotropic polypeptide that can function as a morphogen, motogen, mitogen, angiogen, carcinogen, and tumor suppressor, depending on the target cell and tissue. Previous studies from our laboratory using transgenic mice have shown that HGF gene expression is tightly regulated at the transcriptional level and that the upstream regulatory elements are crucial for the control of HGF gene transcription. In the present study, we have identified and characterized one of these elements as a peroxisome proliferator-activated receptor gamma (PPARgamma)-responsive element. This regulatory element was localized at -246 to -233 base pairs upstream from the transcription start site of the HGF gene promoter having the sequence GGGCCAGGTGACCT. Gel mobility shift and supershift assays demonstrated that this cis-acting element strongly binds to the PPARgamma isoforms as well as to chicken ovalbumin upstream promoter-transcription factor, a member of the orphan nuclear receptor subfamily. Mutational analysis and gel mobility band shift assays indicated that the binding site is an inverted repeat of the AGGTCA motif with two spacers (inverted repeat 2 configuration) and that the two spacers are important for PPARgamma binding. This binding site overlaps with functional binding sites for activating protein-2, nuclear factor 1, and upstream stimulatory factor, and together, they constitute a multifunctional composite binding site through which these different transcription factors exert their regulatory effects on HGF promoter activity. Functional assays revealed that PPARgamma, with its ligand, 15-deoxy-prostaglandin J2, strongly stimulates HGF promoter activity. On the other hand, nuclear factor 1, activating protein-2, and chicken ovalbumin upstream promoter-transcription factor transcription factors repress the stimulatory action of PPARgamma by competing with PPARgamma for their overlapping binding sites. Furthermore, for the first time, our studies demonstrate that the PPARgamma ligand, 15-deoxy-prostaglandin J2, induces endogenous HGF mRNA and protein expression in fibroblasts in culture.

Hepatocyte growth factor is an important multifunctional growth factor whose gene expression is tightly regulated at the transcriptional level. Previous studies from our laboratory have shown that several cis-acting elements are present in the promoter and proximal promoter region of the HGF gene. In this study, we have uncovered that AP2 transcription factor specifically binds to a regulatory site located at −230 to −260 in the upstream region of the HGF gene promoter. Gelshift and supershift assays confirmed that AP2 has high binding affinity to this region. Functional studies which introduced a mutation in the AP2 core binding region as well as cotransfection experiments using an AP2 expression vector revealed that AP2 exerts a repressive role on the HGF gene promoter activity. The AP2 binding site overlaps with those of NF1 and USF/E-box binding sites which we have recently shown to constitute a composite multifunctional docking site for the members of the NF1 and USF transcription factor families. An inverse correlation was noted between AP2 binding activity to this composite site and HGF gene expression in different cell lines. Therefore, AP2-mediated repression of the HGF gene promoter may be part of the molecular mechanism responsible for regulating HGF expression.

Hepatocyte growth factor (HGF) is an important multifunctional cytokine whose gene expression is regulated mainly at the transcriptional level. Previous studies using transgenic mice as well as in vitro analyses showed that a potential regulatory element(s) exists between -260 to -230 bp in the upstream region of the HGF gene promoter. In the present study, we have discovered that this region is a composite site through which members of the nuclear factor 1 (NF1) and upstream stimulatory factor (USF) families bind to and regulate HGF gene transcription. Gel mobility shift and supershift assays revealed that USF and NF1 have high binding affinity for this region and that the binding sites of the two different transcription factor families overlap. Functional studies showed that NF1 suppresses HGF gene promoter activity and that USF has an activating function. We found that the NF1/X and NF1/Red1 isoforms strongly suppressed HGF promoter activity while the NF1/L variant had no obvious effects. USF1, but not USF2, of the USF family stimulated HGF gene promoter activity. More interestingly, during liver regeneration after partial hepatectomy, a process which activates the HGF gene, we noted that the binding activity of USF to the HGF promoter element increased while that of NF1 decreased. These data provide insight into the molecular mechanisms that govern HGF gene transcription. Oncogene (2000).

Hepatocyte Growth Factor (HGF) exerts its biological effects via binding and activating a transmembrane protein tyrosine kinase receptor known as c-Met. Previous studies from our laboratory demonstrated that c-met gene expression is inducible by its own ligand (HGF). However, the molecular mechanism(s) involved in this process are unknown. The present study was carried out to address this question. Transfection of various c-met-CAT promoter constructs into the mouse hepatocellular carcinoma cell line Hepa 1-6 in combination with electrophoretic mobility shift assays (EMSA) identified the responsive element as an activated protein-1 (AP-1) binding site (TGAGTCA) within the c-met core promoter region at position -158 to -152. The c-met AP-1 element binds specifically to AP-1 protein as verified by supershift assays. EMSA studies and mutational analyses of the promoter region also revealed that the members of the Sp family of transcription factors (Sp-1 and Sp-3) bind to the c-met Sp-1 element (located at position -124) which is adjacent to the AP-1 site. We show that Sp binding dampens binding of AP-1 to its cognate site in the c-met promoter region. Stimulation of Hepa 1-6 cells with HGF resulted in a rapid and dramatic enhancement of the AP-1 binding activity as well as an overall increase in the level of AP-1 protein. Cotransfection of AP-1 expression vectors (c-Fos plus c-Jun) with c-met promoter constructs resulted in stimulation of c-met promoter activity. We found that transactivation of the c-met promoter by AP-1 can be blocked by Curcumin, an inhibitor of AP-1. Moreover, we found that the induction of the endogenous c-met gene by HGF is inhibited by the addition of Curcumin. The results demonstrate that the HGF-induced transcription of the c-met gene by HGF is, at least in part, due to activation of the AP-1 pathway.

In the present study, we have investigated the possible involvement of p53 in the transcriptional regulation of the c-met gene. Cotransfection of various c-met promoter reporter vectors with p53 expression plasmids demonstrated that only wild-type p53 but not tumor-derived mutant forms of p53 resulted in a significant enhancement of c-met promoter activity. Functional assays revealed that the p53 responsive element in the c-met promoter region is located at position -278 to -216 and confers p53 responsiveness not only in the context of the c-met promoter but also in the context of a heterologous promoter. Electrophoretic mobility shift assays using purified recombinant p53 protein showed that the p53 binding element identified within the c-met promoter specifically binds to p53 protein. Induction of p53 by UV irradiation in RKO cells that express wild-type p53 increased the level of the endogenous c-met gene product and p21(WAF1/CIP1), a known target of p53 regulation. On the other hand, in RKO cells in which the function of p53 is impaired either by stable transfection of a dominant negative form of p53 or by HPV-E6 viral protein, no induction of the endogenous c-met gene or p21(WAF1/CIP1) was noted by UV irradiation. These results suggest that the c-met gene is also a target of p53 gene regulation.

Hepatocyte growth factor (HGF) is a polypeptide with mitogenic, motogenic, and morphogenic effects on different cell types including hepatocytes. HGF is expressed as two biologically active isotypes resulting from alternative RNA splicing. The roles of each HGF isoform in development, liver regeneration and tumorigenesis have not yet been well characterized. We report the generation and analysis of transgenic mice overexpressing the five amino acid-deleted variant of HGF (dHGF) in the liver by virtue of an albumin expression vector. These ALB-dHGF transgenic mice develop normally, have an enhanced rate of liver regeneration after partial hepatectomy, and exhibit a threefold higher incidence of hepatocellular carcinoma (HCC) beyond 17 months of age. Moreover, overexpression of dHGF dramatically accelerates diethyl-nitrosamine induced HCC tumorigenesis. These tumors arise faster, are significantly larger, more numerous and more invasive than those appearing in non-transgenic littermates. Approximately 90% of female dHGF-transgenic mice had multiple macroscopic HCCs 40 weeks after injection of DEN; whereas the non-transgenic counterparts had only microscopic nodules. Liver tumors and cultured tumor cell lines from dHGF transgenics showed high levels of HGF and c-Met mRNA and protein. Together, these results reveal that in vivo dHGF plays an active role in liver regeneration and HCC tumorigenesis.

The c-met gene encoding Hepatocyte Growth Factor Receptor is predominantly expressed in epithelial cell types and overexpressed in a variety of human and mouse neoplastic tissues and cell lines. To understand the molecular mechanisms of the transcriptional regulation of this gene, we have cloned and functionally characterized the mouse c-met promoter region. Transient transfection analysis using a series of 5'-end deletion met-CAT chimeric constructs in epithelial (C-33A) and fibroblast (NIH3T3) cell lines demonstrated that the c-met promoter acts in a cell-type specific manner. These experiments also localized functionally important regulatory regions at -1390 to -279, relative to the transcription start site, which exert repressive activity, and at -278 to -77 which exhibit enhancing effects on c-met promoter activity. Further analysis by electrophoretic mobility shift assays using specific competitors and antibodies identified Sp1 protein binding to two cognate response elements at -221 and -124 within the enhancer region. Cotransfection experiments revealed that Sp1 stimulated promoter activity of the met-CAT constructs containing the two Sp1 binding sites. These results demonstrate that Sp1 is actively involved in the transcriptional regulation of the c-met promoter.

Met and ron proto-oncogenes encode the cell surface receptors for hepatocyte growth factor (HGF) and hepatocyte growth factor-like (HLP) protein, respectively, and induce mitogenesis, motogenesis, morphogenesis, and metastatic activity in various cell types. Overexpression of met in human carcinoma has been reported by several groups including ours; however, the mechanisms that control met gene expression are thus far unclear. The present study focuses on the expression and regulation of the Met and Ron receptors in human hepatocellular carcinoma (HCC). We report here that abnormal expression of met and ron proteins occurs in some cases of human HCC. Using several HCC cell lines as a model system, we show that HGF, as well as other cytokines, such as epidermal growth factor (EGF), interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha), induce met and ron expression. Using several chimeric constructs consisting of various lengths of the met promoter region fused to the reporter gene of chloramphenicol acetyl transferase (CAT), and by performing transient transfection of these constructs into HepG2 cells, we show that induction of met gene expression by HGF and other cytokines is, at least in part, through up-regulation of met gene promoter activity. The DNA region conferring responsiveness to cytokine induction was located within 0.2 kb of the met core promoter. Interestingly, EGF did not stimulate met promoter activity in any of the met-CAT chimeric constructs. These results provide evidence that met and ron are modulated in the liver by a similar cytokine network. In the case of met expression, the 0.2-kb region in the met gene promoter may play an important role in mediating its gene induction in response to HGF and other cytokines. Our results also suggest that unregulated expression of met and ron may be associated with pathological conditions, such as HCC, in the liver. (Hepatology 1997 Jul;26(1):59-66)

Background/Aims: Vitamin D (D) depletion is a common feature of chronic liver diseases. In past years, disturbances in calcium metabolism involving inadequate D and parathyroid hormone status have been reported to significantly impair the hepatic regeneration process following partial hepatectomy in the rat. The purpose of this study was to investigate how hypocalcemia and D deficiency affect specific cell markers of hepatic compensatory growth. Methods: Steady-state mRNA levels of gene markers of the regeneration process were investigated following 23 partial hepatectomy. The response of hypocalcemic D-depleted rats was compared to that of animals whose calcium status had been normalized by repletion with the active D hormone 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ). Results: The transcript for the major hepatic mitogen HGF increased in both groups after partial liver resection but the increase was significantly lower as well as delayed in livers obtained from calcium deficient rats in the prereplicative phase of the regeneration process. TGFα mRNA levels were also found to be significantly lower in calcium deficient rats at all time-points following partial hepatectomy, while the relative behavior of the tandem TGFα-EGER indicated an early dominant effect in normocalcemic 1,25(OH) 2 D 3 -repleted animals. HGF-c- met mRNA levels also indicated that the 1,25(OH) 2 D 3 -repleted animals reacted more promptly to the regeneration stimuli. Indeed, while relative (1,25(OH) 2 D 3 /D-Ca- ratio) maximum mRNA levels were observed 12 h following liver resection in 1,25(OH) 2 D 3 -treated animals, relative peak levels were only apparent 24 h post-surgery in hypocalcemic rats. Maximum cyclin D 1 (a marker of the G1 phase of the cell cycle) mRNA occurred between 8–18 h after partial hepatectomy in 1,25(OH) 2 D 3 -repleted animals to return to base-line value thereafter, but in hypocalcemic rats the transcript levels remained significantly below 1,25(OH) 2 D 3 -repleted animals during the prereplicative period with increases above initial values between 12–24 h post-surgery. Both cyclin A (an S phase marker) transcripts (1.8 and 2.9 kb) were influenced by the regeneration process. The transcripts significantly and sharply increased in hypocalcemia between 30–36 h following partial hepatectomy to decrease thereafter, while the increase was observed between 24–30 h, and at 48 h (1.8 kb) in 1,25(OH) 2 D 3 -repleted animals. Liver weight recovery was also found to be decreased in D-depleted rats over the 48 h period of observation. Conclusions: Our data further confirm the presence of an impaired regeneration process in hypocalcemia of D deficiency which seems to be associated with gene markers indicating an inefficient transit across the G 1 phase of the cell cycle.

Hepatocyte growth factor (HGF) is a multifunctional cytokine that controls the growth and differentiation of various tissues. Previously, we described the existence of a negative cis-acting regulatory element(s) within the -1- to -0.7-kilobase pair (kb) portion of the 5'-flanking region of the mouse HGF promoter. In the present study, we show that the repressor element is located at position -872 to -860 base pairs and comprises an imperfect estrogen-responsive element 5'-AGGTCAGAAAGACCA-3'. We demonstrate that chicken ovalbumin upstream promoter transcription factor (COUP-TF), a nuclear orphan receptor belonging to the steroid/thyroid hormone receptor superfamily, through binding to this site effectively silences the transcriptional activity of the HGF promoter. We show that estrogen receptor, on the other hand, relieves the repressive action of COUP-TF, resulting in the induction of the HGF promoter. Using mice transgenic for either 2.7 or 0.7 kb of the HGF promoter region linked to the chloramphenicol acetyltransferase reporter gene, we found that injection of estradiol stimulates HGF promoter activity in tissues such as the mammary gland and ovary of mice harboring 2.7 but not 0.7 kb of the mouse HGF promoter region. Potential involvement of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors in the regulation of HGF gene expression is also discussed.

Liver regeneration factor belongs to the leucine-zipper family of transcription factors. It was originally cloned and characterized through differential screening of a regenerating rat liver cDNA library. The mRNA for liver regeneration factor-1 is barely detectable in normal rat liver but is dramatically induced after two-thirds hepatectomy, with a peak 1 to 3 hr after surgery. The nature of the signaling molecule(s) for this rapid induction is not known. It has been suggested that the liver regeneration factor-1 protein product, through complex interactions with other transcription factors such as c-Jun and Jun-B, controls expression of genes that are required during the G1 phase of hepatic growth. Hepatocyte growth factor has been shown to be the most potent mitogen for hepatocytes in vitro and in vivo. Plasma levels of hepatocyte growth factor rapidly (within 30 min) increase after loss of hepatic parenchyma induced by partial hepatectomy or carbon tetrachloride treatment. It has been postulated that hepatocyte growth factor plays a crucial role in stimulating the hepatocyte to enter the cell cycle. In this communication, we report that addition of pure hepatocyte growth factor to primary cultures of rat hepatocytes in the absence of serum and insulin results in rapid and transient induction of liver regeneration factor-1 mRNA (more than 20-fold) with a peak of expression 1 hr after treatment. The levels of jun-B and c-fos mRNAs, which are also known to be induced during the early hours of liver regeneration, were also increased after treatment of isolated hepatocytes with hepatocyte growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatocyte growth factor and transforming growth factor-α are two well-known hepatomitogens for primary hepatocyte cultures. Here we report that these two growth factors also stimulate in vivo DNA syntheses in normal, unoperated, adult rat liver after 24-hr continuous intraportal infusion. As determined by an immunohistochemical staining technique, 5-bromo-2′-deoxyuridine incorporation was increased in a dose-dependent fashion after infusion of up to 10 μg of growth factor/100 gm body weight in the rat. Stimulation of DNA synthesis was seen in the periportal area. Pretreatment using intraportal infusion of collagenase (1 U/kg body weight) for 4 hr before administration of growth factor increased the labeling by 2- to 4-fold to a labeling index range of 48% to 52%. These results suggest that collagenases and possibly other proteases are involved in making hepatocytes competent to respond to growth factors at very early stages of liver regeneration. (HEPATOLOGY 1994;19:1521–1527.)

A cDNA encoding mouse hepatocyte growth factor (HGF) has been cloned and completely sequenced by use of reverse transcriptase-polymerase chain reaction (RT-PCR) and subsequent cloning. Sequence analysis reveals that mouse HGF, similar to its human and rat counterparts, consists of 728 amino acids, and both the alpha- and beta-chains are encoded in a single open reading frame. Strong homology exists in the primary structure of HGF among the three species of mouse, rat and human (more than 90%), especially in Kringle 1 of the alpha chain which is assumed to be an essential domain for binding of HGF to its receptor, c-MET, a proto-oncogene product. Our results suggest the existence of evolutionary pressure to conserve the distinct structure, and presumably the biological functions, of HGF.

Hepatocyte growth factor (HGF) also known as hepatopoietin A (HPTA) (Michalopoulos, FASEB J., 4:176-187, 1990) is a heparin-binding growth factor whose characterization and tissue distribution have been reported elsewhere. This growth factor was recently cloned and its amino acid sequence determined under the name of hepatocyte growth factor (HGF) (Miyazawa et al., Biochem. Biophys. Res. Commun., 163:967-973, 1989; Zarnegar et al., Biochem. Biophys. Res. Commun., 163:1370-1376, 1989; Nakamura et al., Nature, 342:440-443, 1989). Human placenta is one of the tissues that contains significant amounts of HGF. We isolated HGF from human placenta and characterized its biologic effect on rat hepatocytes. Human placenta HGF was isolated in high purity as a single chain molecule. Single chain HGF stimulated DNA synthesis in primary rat hepatocyte cultures in serum-free medium. The maximal effect was seen at 5-10 ng/ml. The maximal response occurred at 25-48 hours after plating of the hepatocytes. Protein synthesis was also stimulated by HGF in primary rat hepatocyte cultures. There were peak responses at 19-24 and 37-42 hours after plating of the hepatocytes. TGF beta 1 inhibited more than 95% of HGF-induced DNA synthesis but only 25% of HGF-induced protein synthesis. HGF interacted in an additive manner with EGF, a well-known hepatocyte mitogen. There was not an additive interaction between HGF and aFGF. Regenerating liver hepatocytes obtained from rats which underwent two-thirds partial hepatectomies (PHX) also responded to HGF in a dose-dependent manner as the hepatocytes from normal liver.

Hepatocyte growth factor is a protein growth factor with a strong mitogenic effect on hepatocytes. Recently, hepatocyte growth factor and hepatocyte growth factor messenger RNA have been extracted from several organs of humans, rats and rabbits. This study was undertaken to comprehensively define and compare the cellular localization of hepatocyte growth factor in human and rat tissues in detail. Paraffin-embedded sections and frozen sections were examined by immunohistochemistry using a polyclonal antiserum to hepatocyte growth factor. The distribution of hepatocyte growth factor was almost identical in humans and rats. Strong or moderate cytoplasmic immunoreactivity for hepatocyte growth factor was present in most surface epithelia, distal tubules and collecting ducts of the kidneys, large neurons, megakaryocytes, granulocytes, exocrine pancreas, salivary glands, prostate, epididymis and trophoblast. Varying degrees of immunoreactivity were observed in endothelial cells, chondrocytes and macrophages. We conclude that hepatocyte growth factor is widely distributed in numerous tissues and cell types independent of their regenerative activity. This suggests that hepatocyte growth factor may have mitogenic and/or trophic effects on multiple cell types in addition to hepatocytes.

Hepatocyte Growth Factor (HGF) is a potent complete mitogen for primary cultures of hepatocytes in vitro. There is strong evidence that this novel growth factor may mediate hepatocyte regeneration after liver damage. We have shown previously that the amount of immunoreactive HGF markedly increases in the serum of rats soon after partial hepatectomy or CCl4 administration. In the present paper, we demonstrate that the level of HGF mRNA in rat liver also dramatically increases from 3 to 6 hours post hepatectomy, peaks at 12 hr and gradually returns to undetectable levels by 72 to 96 hours post hepatectomy. In separate experiments, DNA synthesis (in vivo) was determined in rat liver remnants after partial hepatectomy. DNA synthesis peaked 24 hr after hepatectomy, 12 hr after the peak of HGF mRNA expression. These results suggest that HGF may be one of the major early signals that triggers hepatocyte proliferation during liver regeneration.

An enzyme-linked immunosorbent assay was used to measure the level of hepatocyte growth factor in rat plasma at various times after two-thirds partial hepatectomy or CCl4 administration. An initial 17-fold rise and 13-fold rise in the level of hepatocyte growth factor was observed 2 hr after partial hepatectomy and CCl4 treatment, respectively, well before the onset of DNA synthesis in the liver. The peaks of DNA synthesis in remnant livers and livers exposed to CCl4 occurred at 24 hr and 48 hr, respectively, as determined by 5-bromo-2'-deoxyuridine labeling and [3H]thymidine uptake by the liver. A later peak level (17-fold above control) of hepatocyte growth factor at 24 hr after CCl4 treatment coincided with strong immunostaining of damaged or necrotic hepatocytes around central veins with an antibody to hepatocyte growth factor. This suggests a later intrahepatic origin of the signals for liver regeneration after hepatotoxic injury subsequent to the early extrahepatic production of hepatocyte growth factor at 2 hr after CCl4 administration. The absence of staining in the liver remnants in partially hepatectomized rats implies that the increase in hepatocyte growth factor seen in the plasma is caused by production at extrahepatic site(s). Possible sources include the pancreas, brain, thyroid and salivary glands, and Brunner's glands of the duodenum. Norepinephrine also increases in plasma as early as 2 hr after hepatectomy. In vitro, [3H]thymidine incorporation into hepatocyte DNA in the presence of hepatocyte growth factor is greater if 10(-5) mol/L norepinephrine is also present in the media.(ABSTRACT TRUNCATED AT 250 WORDS)

The HGF gene is transcriptionally silenced in normal differentiated breast epithelial cells, but its repression fails to occur in mammary carcinoma tissues and cell lines. The molecular mechanisms underpinning aberrant HGF expression in breast cancer cells are unknown. Here we report the discovery of a DNA element located 750 bp upstream from the transcription start site in the human HGF promoter that acts as a transcriptional repressor and is a target of deletion mutagenesis in human breast cancer cells and tissues. This HGF promoter element consists of a mononucleotide repeat of 30 deoxyadenosines (30As), which we have termed "deoxyadenosine tract element" (DATE). Functional studies revealed that truncation mutations within DATE have profound local and global effects on the HGF promoter region by modulating chromatin structure and DNA-protein interactions, leading to constitutive activation of the HGF promoter in human breast carcinoma cell lines. We found that 51% of African Americans and 15% of individuals of mixed European descent with breast cancer harbor a truncated DATE variant (25As or fewer) in their breast tumors and that the truncated allele is associated with cancer incidence and aberrant HGF expression. Notably, breast cancer patients with the truncated DATE variant are substantially younger than those with a wild-type genotype. We also suggest that DATE may be used as a potential genetic marker to identify individuals with a higher risk of developing breast cancer.

Glutathione peroxidase 3 is a selenium-dependent enzyme playing a critical role in detoxifying reactive oxidative species and maintaining the genetic integrity of mammalian cells. In this report, we found that the expression of glutathione peroxidase 3 (GPx3) was widely inactivated in prostate cancers. Complete inactivation of GPx3 correlates with a poor clinical outcome. Deletions (hemizygous and homozygous) of GPx3 gene are frequent in prostate cancer samples, occurring in 39% of the samples studied. The rate of methylation of the GPx3 exon 1 region in prostate cancer samples reaches 90%. Overexpression of GPx3 in prostate cancer cell lines induced the suppression of colony formation and anchorage-independent growth of PC3, LNCaP, and Du145 cells. PC3 cells overexpressing GPx3 reduced invasiveness in Matrigel transmigration analysis by an average of 2.7-fold. Xenografted PC3 cells expressing GPx3 showed reduction in tumor volume by 4.8-fold, elimination of metastasis (0/16 versus 7/16), and reduction of animal death (3/16 versus 16/16). The tumor suppressor activity of GPx3 seems to relate to its ability to suppress the expression of c-met. The present findings suggest that GPx3 is a novel tumor suppressor gene. [Cancer Res 2007;67(17):8043–50]

The death receptor pathway is coupled to the mitochondria apoptosis pathway. However, mitochondrial participation, which is stimulated by Bid but suppressed by Bcl-2/Bcl-xL, is required in certain cells (Type II), but not in others (Type I). While these differences were originally characterized in the lymphoid cell lines, the typical Type II cells are represented by hepatocytes in vivo. The molecular mechanisms that distinguish Type II from Type I cells and the regulation are not fully understood. Fas can be sequestered by the HGF receptor c-Met and high doses of HGF can promote cell death by freeing Fas from c-Met complex. We thus reasoned that treatment of the Type II cells with high doses of HGF could enhance Fas-mediated apoptosis and spare the mitochondria amplification. Indeed, such treatment led to increased apoptosis in Type II lymphoid cells, which could not be blocked by Bcl-xL. Moreover, significant hepatocyte apoptosis was induced by this scheme in the absence of Bid with increased dissociation of Fas from c-Met. These findings indicate that high doses of HGF could be used to promote apoptosis in Type II cells bypassing the requirement for mitochondria activation. J. Cell. Physiol. 213: 556–563, 2007. © 2007 Wiley-Liss, Inc.

Background & Aims Genomic instability promotes colon carcinogenesis by inducing genetic mutations, but not all genes affected by this process have been identified. We investigated whether genomic instability in human colorectal cancer (CRC) cells produces mutations in the hepatocyte growth factor ( HGF ) gene. Methods We genotyped human colon tumor tissues and adjacent nontumor tissues collected from 78 patients University of Pittsburgh Health Sciences and Veterans Hospital, along with 40 human CRC and adjacent nontumor tissues in a commercial microarray. We used cellular, biochemical, and molecular biological techniques to investigate the factors that alter HGF signaling in colon cancer cells and its effects on cell proliferation and survival. Results All tested human CRC tissues and cell lines that had microsatellite instability contained truncations in the regulatory deoxyadenosine tract element (DATE) of the HGF gene promoter. The DATE was unstable in 14% (11 of 78) of CRC samples; DATE truncation was also polymorphic and detected in 18% (13 of 78) of CRC tissues without microsatellite instability. In CRC cell lines, truncation of DATE activated expression of HGF, resulting in its autocrine signaling via MET. This promoted cell proliferation and resistance to necroptosis. HGF signaling via MET reduced levels of the receptor-interacting serine-threonine kinase 1, a mediator of necroptosis, in CRC cells. High levels of HGF protein in tumor tissues correlated with lower levels of receptor-interacting serine-threonine kinase 1 and shorter survival times of patients. Conclusions Thirty-one percent of CRC samples contain alterations in the DATE of the HGF promoter. Disruption of the DATE increased HGF signaling via MET and reduced levels of receptor-interacting serine-threonine kinase 1 and CRC cell necroptosis. DATE alteration might be used as a prognostic factor or to select patients for therapies that target HGF−MET signaling.

Human beta2-glycoprotein I (beta2GPI), also known as apolipoprotein H, has been implicated in haemostasis and the production of anti-phospholipid antibodies. There is a wide range of interindividual variation in beta2GPI plasma levels that is thought to be under genetic control, but its molecular basis remains unknown. To understand the genetic basis of beta2GPI variation, we analyzed the 5' flanking region of the beta2GPI gene for mutation detection by DHPLC and identified a point mutation at the transcriptional initiation site (-1C--A) with a carrier frequency of 12.1%. The mutation was associated with significantly lower beta2GPI plasma levels (P0.0001) and low occurrence of anti-phospholipid antibodies in lupus patients (4.8% antibody-positive group vs. 16.6% in the antibody-negative group; P = 0.019). Northern blot analysis confirmed that the -1C--A mutation was associated with lower mRNA levels and it reduced the reporter (luciferase) gene expression by twofold. Electrophoretic gel mobility shift assay (EMSA) revealed that the -1C--A mutation disrupts the binding for crude hepatic nuclear extracts and purified TFIID. These results suggest that the substitution of C with A at the beta2GPI transcriptional initiation site is a causative mutation that affects its gene expression at the transcriptional level and ultimately beta2GPI plasma levels and the occurrence of anti-phospholipid antibodies.

Hepatocyte growth factor (HGF) and Wnt signaling pathways have been shown to be important in embryogenesis and carcinogenesis. The aim of this study was to elucidate the mechanism of functional similarities observed in the two pathways. We used normal rat liver, primary hepatocyte cultures and a dominant-negative Met expression system to study the effect of HGF on Wnt pathway components. We demonstrate novel association of beta-catenin and Met, a tyrosine kinase receptor of HGF, at the inner surface of the hepatocyte membrane. HGF induces dose-dependent nuclear translocation of beta-catenin in primary hepatocyte cultures that is Wnt independent. The source of beta-catenin for translocation in hepatocytes is the Met-beta-catenin complex, which appears to be independent of the E-cadherin-beta-catenin complex. To test the functionality of this association, we used a dominant-negative Met expression system that expresses only the extracellular and transmembrane regions of the beta-subunit of Met. A loss of Met-beta-catenin association resulted in abrogation of nuclear translocation of beta-catenin upon HGF stimulation. This event is tyrosine phosphorylation dependent, and the association of Met and beta-catenin is crucial for this event. We conclude that the HGF causes similar redistribution of beta-catenin as Wnt-1 in the hepatocytes and that this effect is attributable to subcellular association of Met and beta-catenin. The intracellular kinase domain of Met is essential for tyrosine phosphorylation and nuclear translocation of beta-catenin. Part of the multifunctionality of HGF might be attributable to nuclear beta-catenin and the resulting target gene expression.

Death receptors such as Fas are present in a variety of organs including liver and play an important role in homeostasis. What prevents these harmful receptors from forming homooligomers, clustering, and initiating the apoptotic pathway is not known. Here, we report the discovery of a cell survival mechanism by which Met, a growth factor receptor tyrosine kinase, directly binds to and sequesters the death receptor Fas in hepatocytes. This interaction prevents Fas self-aggregation and Fas ligand binding, thus inhibiting Fas activation and apoptosis. Our results describe a direct link between growth factor tyrosine kinase receptors and death receptors to establish a novel paradigm in growth regulation.

Liver selected B16-LS9 melanoma cells show a dramatic overexpression of the proto-oncogene c-met, the cellular receptor for hepatocyte growth factor/scatter factor. As a consequence, c-met becomes constitutively active, and the cells become more responsive to hepatocyte growth factor stimulation. We have investigated the molecular mechanisms regulating c-met expression in both the parental line B16-F1, which has low expression levels, and the liver-specific B16-LS9, overexpressing c-met. Overexpression is observed at the protein and mRNA levels, however without further evidence of gene amplification or rearrangement. c-met promoter activity was higher in B16-LS9 than B16-F1 cells, and also a nuclear run-off showed higher transcription levels in B16-LS9 cells. Moreover, we found that c-met mRNA had a longer half-life in B16-LS9 cells, thus indicating also the involvement of post-transcriptional regulation mechanisms. Finally, we found evidence that autonomous activation of the melanocortin receptor-1 (MCR-1) is at least partially responsible for c-met upregulation in B16-LS9 cells, since treatment of the cells with a potent MSH antagonist (the agouti peptide) has strong down-regulatory effects. J. Cell. Biochem. 81:477–487, 2001. © 2001 Wiley-Liss, Inc.

To understand the molecular mechanisms of hepatocyte growth factor (HGF) gene transcription in vivo, we report the generation and characterization of transgenic mice harboring various lengths of the mouse HGF promoter linked to the chloramphenicol acetyltransferase reporter gene. Analysis of different tissues of the transgenic mouse lines having the 2.7-kilobase (kb) promoter construct revealed a pattern of reporter gene expression in embryonic and adult tissues that paralleled that of endogenous HGF gene expression. A similar expression pattern was observed in the 0.7-kb transgenic lines. However, in contrast to in vitro data, no promoter activity was detected in four independent transgenic lines harboring the 0.1-kb construct. Akin to the activity of the endogenous HGF gene, which is induced in the liver, lung, and spleen in response to 70% partial hepatectomy, the reporter gene driven by the 2.7-kb promoter construct was strongly induced, whereas that driven by the 0.7-kb promoter construct was modestly induced in these organs after partial hepatectomy. Together, these data suggest that the region between -0.1 and -0.7 kb of the HGF gene promoter is essential to drive its expression in vivo and that additional upstream sequences located between -0.7 and -2.7 kb are also necessary for its maximum inducibility in response to cues that stimulate tissue growth and regeneration.

In this study, we have localized an enhancer element in the 5′-flanking region of the HGF gene promoter and have identified its functional transcriptional factors. Through transient transfection of NIH3T3 fibroblast cells and gel mobility shift assays, the functional binding site was localized to a short region (−318 to −303 bp from the transcription start site) which has a CTCCC sequence. This motif is also conserved in the human HGF promoter and acts as a binding site for the Sp family of transcription factors. In the presence of NIH3T3 nuclear protein extracts, this enhancer region formed specific DNA-protein complexes which were totally abrogated by excess amounts of consensus Sp1 oligonucleotide. In gel mobility supershift assays, anti-Sp1 and anti-Sp3 antibodies specifically recognized these complexes as was evident by their slower migration. Site-specific mutation of this binding motif resulted in total loss of Sp1 and Sp3 binding and a concomitant loss of enhancer function within the context of the HGF promoter. Furthermore, in transient cotransfection assays, overexpression of Sp1 and/or Sp3 stimulated HGF promoter activity independently and additively through binding to the Sp1 binding site in the HGF gene promoter region. DNaseI hypersensitive analysis of freshly isolated nuclei from NIH3T3 cells revealed that five hypersensitive sites (HSS) are present within the 2 kb region immediately upstream of the HGF promoter. One of these sites was mapped to position −0.3 kb from the transcription start site. These data show that both Sp1 and Sp3 transcription factors upregulate HGF promoter activity by binding to the Sp1 binding site at position −318 to −303 bp in the HGF gene promoter.