A utilização de fatores hepatotróficos (FH) tem trazido importantes avanços no tratamento de algumas doenças hepáticas. A avaliação dos efeitos dessas substâncias pode ser feita com o uso de modelos in vivo, como a regeneração hepática após a hepatectomia parcial ou in vitro, como a cultura de hepatócitos, células estreladas ou outros tipos celulares do fígado. O modelo de cultura demonstra ser útil por possibilitar a análise individualizada de determinadas substâncias ou soluções diretamente nas células-alvo, facilitando o delineamento de seu mecanismo de ação. Dessa forma, o presente trabalho estudou in vitro os efeitos da administração de fatores hepatotróficos (FH) em hepatócitos isolados de fígados de ratos, avaliando seu metabolismo e proliferação celular. Trinta ratos Wistar fêmeas foram utilizados, o fígado retirado e, por digestão enzimática in situ, suas células foram dissociadas e os hepatócitos separados em gradiente de Percoll 45%; cultivados em meio DMEM/F12, tratadas com diferentes concentrações dos FH (1X, 5X e 10X) e analisadas em intervalos de 24, 48 e 72 horas. A viabilidade e a proliferação celular foram avaliadas pelo método colorimétrico MTT, a toxicidade pelo iodeto de propídeo, o potencial elétrico da membrana mitocondrial pela Rodamina 123 e a expressão da citoqueratina 8, 18 e desmina como marcadores do citoesqueleto. O metabolismo hepático foi avaliado pela depuração do verde de indocianina (VIC), pela quantificação de colágeno tipo I e pela formação de radicais lipídicos poliinsaturados peroxidados. A técnica utilizada para a obtenção de hepatócitos mostrou-se eficaz com viabilidade superior a 90%, sem apresentar toxicidade, com manutenção do potencial mitocondrial e com marcação positiva para citoqueratinas 8, 18 e desmina. A adição dos FH aumentou a proliferação dos hepatócitos nos três períodos analisados em relação ao grupo controle. Os FH não demonstraram toxicidade em cultura primária de hepatócitos em nenhuma das concentrações avaliadas ao longo de todos os períodos experimentais. A adição dos FH na concentração de 10X evitou a formação de radicais livres, protegendo os hepatócitos da lipoperoxidação. Por outro lado, a avaliação funcional das culturas primárias pelo teste VIC mostrou-se eficaz na determinação do metabolismo dos hepatócitos, como a manutenção dos níveis das transaminases e amilase. Os hepatócitos mantidos em cultura primária após a adição dos FH em todos os períodos analisados aumentaram a produção de colágeno, e também foram capazes de modificar a distribuição da população de células nas fases quiescentes, aumentando sua capacidade de síntese. A adição dos FH nas concentrações estudadas nas culturas de hepatócitos mostrou-se eficaz na manutenção dessas células, mantendo-se funcionalmente ativos, com a expressão dos marcadores de seu metabolismo e diferenciação. / The use of hepatrotophic hic factors (HF) has provided important advances in the treatment of several hepatic disorders. The evaluation of this treatment may be carried out by in vivo models, as experiments on hepatic regeneration after partial hepatectomy; or in vitro models as culture of hepatocytes, stellate cells or any other hepatic cell. This last model allows an individual analysis of the studied substances on their target cells, providing the possibility of further investigation on the mechanisms involved. Therefore, the present study evaluated the in vitro effects of the hepatotrophic factors administration on the metabolism and proliferation of cultured hepatocytes from normal rat liver. Liver from 30 Wistar rats were used, and after in situ enzimatic dissociation, hepatocytes were collected and separated by 45% Percoll density gradient, cultivated in DMEN/F12 media supplemented with different HF concentrations (1×, 5× and 10×) and finally analyzed at 24, 48 and 72hs intervals. Cell viability and proliferation were assessed by MTT colorimetric assay, cell toxicity by propidium iodide, mitochondrial membrane electrical potential by 123 rodamine test and cytokeratin 8, 18 and desmin as cytoskeleton markers. Hepatic metabolism was evaluated by infusion of indocianine green (ICG), by type I collagen quantification and by peroxided polyunsaturated lipid radicals production. The techniques used in order to collect hepatocytes proved to be efficient, with a viability higher than 90%, no toxicity, and the maintenance of the mitochondrial membrane potential and positive labeling for cytokeratins (CK) 8, 18 and desmin. The HF addition increased the proliferation of hepatocytes in the three periods analyzed, when compared to the control group. The HF did not show any toxicity in primary hepatocytes culture regardless of the concentration evaluated along the experimental periods. The HF addition impaired the production of free radicals, protecting the hepatocytes from the lipid peroxidation. On the other hand, the functional evaluation of primary hepatocytes cultures using the ICG test proved to be efficient in the determination of the hepatocytes metabolism, as the maintenance of the transaminase and amylase levels. The hepatocytes kept in primary culture after HF addition in all the analyzed periods increased the collagen production and were also able to shift the distribution of quiescent cells population of, thus increasing their synthesis capacity. The HF addition in the studied concentrations in hepatocytes cultures proved to be efficient in the maintenance of these cells, that remain functionally active and expressing their metabolism and differentiation characteristic markers.

L'incidence des maladies hépatiques est en augmentation, ce qui rend urgente la recherche de systèmes de vectorisation d'agents thérapeutiques vers le foie. La maladie sur laquelle porte notre étude est la maladie orpheline de Wilson. Elle induit une accumulation de cuivre (Cu) principalement dans les hépatocytes et les chélateurs de Cu systémiques utilisés à ce jour pour la traiter présentent de nombreux effets secondaires. C'est dans ce contexte que nous proposons une stratégie innovante qui consiste à piéger sélectivement le cuivre en excès dans les cellules hépatiques. Des chélateurs sélectifs du Cu(I), degré d'oxydation du Cu disponible intracellulaire, ont été conçus. Afin de cibler ces derniers vers les cellules hépatiques, nous avons focalisé notre intérêt sur le récepteur aux asialoglycoprotéines (ASGP-R), une lectine abondamment et presque exclusivement exprimée à la membrane des hépatocytes.Deux familles de glycoconjugués contenant une unité de ciblage associée à une unité de vectorisation des hépatocytes ont été développées : l'une possède une plateforme cyclodécapeptidique et l'autre une plateforme pseudopeptidique d'architecture tripodale. Les unités de ciblage sont composées de N-acétylgalactosamines présentés de manière multivalente, permettant une reconnaissance et une endocytose sélective des glycoconjugués dans les hépatocytes par l'intermédiaire de la lectine ASGP R. Les deux familles de composés ont montré leur capacité à entrer dans les cellules hépatiques et à faire diminuer le taux de Cu intracellulaire disponible.L'objectif de ce travail de thèse est l'étude et l'optimisation du système de ciblage. Les efficacités d'internalisation des deux familles de glycoconjugués dans les hépatocytes sont pour cela comparée. En outre, l'influence de la structure moléculaire des glycoconjugués sur l'efficacité de ciblage est étudiée. Plusieurs glycoconjugués appartenant à chacune des deux familles de composés ont donc été synthétisés, et leur propriétés d'internalisation dans les hépatocytes humains analysée par cytométrie en flux. / Regarding the increasing incidence of liver diseases in the past decades, the discovery of drug delivery systems is becoming a major research area. In particular, the Wilson disease is a liver dysfunction, which needs more specific treatments than those available nowadays. This genetic disorder induces a toxic copper overload in the hepatocytes. Because current copper chelating therapies present many side effects due to their lack of specificity, we propose an innovative strategy that would selectively detoxify copper in liver cells. Since excess intracellular Cu is in the +I oxidation state, we figured that a chelator that would enter the hepatocytes and be specific for Cu(I) could represent an efficient strategy. To selectively target the hepatic cells, we focus our interest on a lectin, which is highly and exclusively expressed in the membrane of the hepatocytes: the asialoglycoprotein receptor (ASGP-R).Two different kinds of glycoconjugates that contain a Cu(I) chelating unit associated to liver targeting systems have been obtained : one has a cyclodecapeptide scaffold and the other a tripodal pseudopeptide core. The targeting units are composed of N-acetylgalactosamine (GalNAc) residues multivalently presented, allowing the selective recognition and endocytosis of the glyconconjugates thanks to ASGP-R. These two families of molecules were demonstrated to enter hepatocytes and to chelate intracellular copper.The aim of the present work is to study and optimise the targeting system. We have compared the efficiency of the targeting systems of the two families of molecules. Moreover the influence of several structural parameters on the internalisation efficiency was determined. Several compounds belonging to cyclodecapeptide and tripode families have been synthesised, and their ability to enter in human hepatocytes has been analysed by flow cytometry.

Siu, Yeung Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 65-75). / Abstracts in English and Chinese. / Title Page --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iii / Acknowledgement --- p.iv / Table of Contents --- p.v-viii / List of Figures --- p.ix / List of Tables --- p.x / Abbreviations --- p.xi-xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Growth factors involved in hepatocytes proliferation --- p.1-6 / Chapter 1.1.1 --- Hepatocyte growth factor (HGF) --- p.1 / Chapter 1.1.2 --- Tumor necrosis factor-a (TNF-α) and interleukin-6 (IL-6) --- p.2 / Chapter 1.1.3 --- Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) --- p.3 / Chapter 1.1.4 --- Other comitogens --- p.4 / Chapter 1.1.5 --- Transforming growth factor-β (TGF-β) --- p.5 / Chapter 1.2 --- Bone marrow stromal cells and hepatocytes proliferation --- p.7-12 / Chapter 1.2.1 --- Role of bone marrow stromal cells in bone marrow --- p.7 / Chapter 1.2.2 --- Bone marrow as a source of hepatic oval cells --- p.8 / Chapter 1.2.3 --- Growth factors secreted by bone marrow stromal cells involved in hepatocytes proliferation --- p.9 / Chapter 1.2.4 --- Endocrine in hepatocytes proliferation --- p.12 / Chapter 1.3 --- Objective of this study --- p.13-15 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Cell cultures --- p.16 / Chapter 2.2 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.17-18 / Chapter 2.2.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.17 / Chapter 2.2.2 --- "Incubation of serum deprived Hep 3B, Hep G2, C3A, SK- Hep-1 and Chang cells with mitogenic stimuli" --- p.17 / Chapter 2.2.3 --- Cell cycle analysis by flow cytometry using propidium iodide staining --- p.17 / Chapter 2.3 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.18-20 / Chapter 2.3.1 --- Partially growth arrested human SK-Hep-1 cells --- p.18 / Chapter 2.3.2 --- Human bone marrow stromal cells --- p.19 / Chapter 2.3.2.1 --- Bone marrow stromal cellular extract --- p.19 / Chapter 2.3.2.2 --- Total protein assay --- p.19 / Chapter 2.3.3 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extracts --- p.20 / Chapter 2.4 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.21-22 / Chapter 2.4.1 --- Dialysis --- p.21 / Chapter 2.4.2 --- Temperature treatment --- p.21 / Chapter 2.4.3 --- Proteolysis --- p.22 / Chapter 2.5 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.22-26 / Chapter 2.5.1 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.22 / Chapter 2.5.2 --- Metabolic labeling of SK-Hep-1 cells with [32P]orthophosphate --- p.23 / Chapter 2.5.3 --- Incubation of labeled SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.23 / Chapter 2.5.4 --- SK-Hep-1 cells lysate extraction --- p.23 / Chapter 2.5.5 --- Two-dimensional electrophoresis --- p.24 / Chapter 2.5.5.1 --- First dimension isoelectric focusing --- p.24 / Chapter 2.5.5.2 --- Second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis --- p.25 / Chapter 2.5.6 --- Amplification of radiolabeled signal by EN3HANCE --- p.25 / Chapter 2.5.7 --- Visualization of autoradiography --- p.26 / Chapter 2.5.8 --- Visualization by silver staining --- p.26 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.27-30 / Chapter 3.1.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.27 / Chapter 3.1.2 --- DNA synthesis of hepatic cell lines in response to 10 % FBS after serum deprivation --- p.29 / Chapter 3.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.31-39 / Chapter 3.2.1 --- Cell cycle distribution of partially growth arrested SK-Hep-1 cells in response to mitogens --- p.31 / Chapter 3.2.2 --- Time course on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to FBS and bone marrow stromal cellular extract --- p.36 / Chapter 3.2.3 --- Dose response on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to bone marrow stromal cellular extracts --- p.38 / Chapter 3.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.40-44 / Chapter 3.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.45-49 / Chapter 3.4.1 --- Mitogenic response of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.45 / Chapter 3.4.2 --- Early intracellular signaling of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.47 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.50 / Chapter 4.2 --- "Mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.51 / Chapter 4.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.52 / Chapter 4.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.53 / Chapter 4.5 --- Possible directions for future investigation --- p.55 / Chapter 4.6 --- Conclusions --- p.56 / Chapter Chapter 5 --- Appendices / Chapter 5.1 --- Reagents and solutiuons --- p.57-64 / Chapter 5.1.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.57 / Chapter 5.1.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.59 / Chapter 5.1.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.60 / Chapter 5.1.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.61 / Chapter Chapter 6 --- References --- p.65-75

Plusieurs données expérimentales suggèrent que la protéine Core du virus de l’Hépatite B (HBV), en plus de ses fonctions structurales pour la formation des nucléocapsides dans le cytoplasme, pourrait avoir des fonctions régulatrices importantes dans le noyau des hépatocytes infectés. En effet, Core s’associe à l’ADNccc et aux promoteurs de certains gènes cellulaires dans le noyau des hépatocytes infectés et pourrait ainsi contrôler leur régulation transcriptionnelle. De plus, de par sa capacité à lier les ARN, elle pourrait également participer au métabolisme post-transcriptionnel de gènes viraux et/ou cellulaires. Pour caractériser ces fonctions, nous avons réalisé une analyse protéomique des facteurs cellulaires qui interagissent avec la protéine Core dans le noyau d’hépatocytes humains. Cet interactome a mis en évidence un grand nombre de protéines de liaison aux ARN (RBP), qui participent au métabolisme des ARN et en particulier aux mécanismes d’épissage. Deux interactants majeurs de Core ont été plus particulièrement étudiés, SRSF10 et RBMX, impliqués notamment dans l’épissage et la réparation de l’ADN. Une analyse fonctionnelle effectuée par une approche siRNA a montré que SRSF10 et RBMX affectent différemment le niveau des ARN viraux, vraisemblablement en agissant à des étapes différentes du cycle viral. De même, un composé ciblant l’activité de certaines RBP diminue fortement la réplication d’HBV en affectant l’accumulation des ARN viraux. Ainsi, ces résultats suggèrent que Core pourrait interagir avec certaines RBP pour contrôler le destin des ARN viraux et/ou cellulaires, une piste intéressante pour le développement de nouvelles stratégies antivirales ciblant l’hôte / Converging evidences suggest that the Hepatitis B virus (HBV) core protein, beside its well-known structural role to form nucleocapsids in the cytoplasm, could have important regulatory functions in the nucleus of infected hepatocytes. Indeed, nuclear Core was shown to associate with the cccDNA and to the promoters of some cellular genes, suggesting that Core may control viral and/or cellular gene expression. In addition, Core has the capacity to bind RNA, and may thus regulate HBV RNA metabolism. To elucidate these functions, we performed a proteomic analysis of the cellular factors interacting with nuclear Core in human hepatocytes. This interactome revealed a majority of highly interconnected RNA-binding proteins (RBPs), which participate in several steps of mRNA metabolism, including transcription, splicing and nuclear egress. We focused on two major Core-interacting factors, SRSF10 and RBMX that were previously involved in splicing and DNA repair. Functional analyses performed by a siRNA approach indicated that RBMX and SRSF10 were able to differentially regulate the levels of all viral RNAs most likely by acting at different steps of the viral life-cycle. Similarly, a small compound, affecting the activity of selected RBPs, severely impaired HBV replication by strongly reducing viral RNA accumulation. Altogether, these results strongly suggest that Core interacts with some selected RBPs to control the fate of viral and/or cellular RNAs and provide new critical information for the development of novel host-targeting antiviral agents (HTA)

Malaria is a critical and global public health problem, affecting over 200 million people every year, resulting in over 500,000 deaths. A vaccine is not currently available and only one drug, primaquine, is effective against the dormant stages of Plasmodium vivax. Preclinical assessment of novel therapeutic drugs and vaccines is hampered by the lack of an in vitro liver model for P. falciparum and P. vivax. To provide a stable human hepatocyte-based culture platform for parasite development, we engineered a microfluidic bilayer device capable of both simple and complex culture methods, including perfusion and co-culture, to better understand the requirements of both hepatocytes as host cells and parasite liver stage development. As only the mechanical compaction of the device channels was found necessary for stable hepatocyte culture, and only a validated host cell lot was found necessary for improved parasite development rates, we present an efficient and simple Plasmodium liver model capable of supporting P. vivax dormant forms. Device liver platforms were used to generate kill curves of P. vivax and P. falciparum after three days under primaquine drug pressure. Furthermore, an anti-CSP antibody-based inhibition of development assay with P. falciparum successfully demonstrated antibody-specific liver stage inhibition. Ongoing studies aim to identify the minimal unit of the culture system necessary to be multiplexed in a fully functional and efficient drug and vaccine discovery platform.

The influence of hyperlipidemia on the pharmacodynamic and pharmacokinetic aspects of lipophilic drugs was explored. The antiarrhythmic, amiodarone, and the antifungal, ()-ketoconazole, were used as model drugs. Experimental hyperlipidemia was induced in rat using poloxamer 407 and two sensitive novel HPLC assays were developed. In a multiple dosing study, hyperlipidemia increased amiodarone plasma concentrations, heart concentrations and electrocardiographic changes. The amiodarone heart uptake could not be totally attributed to its unbound fraction, where the cardiac very low density lipoprotein receptors seemed to play a role in the uptake of bound drug. Amiodarone liver metabolism in presence and absence of hyperlipidemia was studied using isolated primary rat liver hepatocytes. The metabolism of amiodarone was lower in hepatocytes isolated from hyperlipidemic than those from normolipidemic rats. Hyperlipidemic serum resulted in a decrease in amiodarone metabolism and when coincubated, the expected decrease in unbound fraction seemed to resulted in greater inhibition of metabolism. ()-Ketoconazole showed stereoselectivity in its pharmacokinetics in rat with (+)-ketoconazole showing higher plasma concentrations than its antipode. This was attributed to its higher protein binding. There was no difference in the total bioavailability of the two enantiomers. Ketoconazole enantiomers exhibited nonlinear pharmacokinetics. In normolipidemic rat plasma ketoconazole enantiomers were more than 95% bound to lipoprotein deficient fraction. Hyperlipidemia resulted in shifting both enantiomers 20% to very low density and low density lipoprotein fractions. In a pharmacokinetic assessment, hyperlipidemia was found to increase ketoconazole enantiomer volume of distribution. Moreover, the stereoselectivity ratios of most pharmacokinetic parameters were changed. After oral dosing, the uptake of (-)-ketoconazole was significantly decreased. Since ketoconazole is used as a potent CYP3A inhibitor, alteration in liver concentrations of (-)-ketoconazole, the more potent inhibitory enantiomer, could decrease its CYP inhibitory potential. Hyperlipidemia potentiated the CYP-mediated interaction between ketoconazole and midazolam with significantly higher midazolam AUC and lower clearance. This was attributed to the inhibitory action of ketoconazole and the effect of hyperlipidemia on the binding of midazolam. Hyperlipidemia was found to unexpectedly decrease midazolam unbound fraction in plasma. In conclusion, the findings could explain some unexpected dose versus effect outcomes in hyperlipidemic patients receiving amiodarone or ketoconazole. / Pharmaceutical Sciences

In the past, hyperlipidemia (HL) has been shown to affect the pharmacokinetic and pharmacodynamic properties of lipophilic drugs extensively bound to lipoproteins, including halofantrine (HF). The present thesis examined the effect of HL on the biodistribution, metabolism and electrocardiographic (ECG) effects of HF in the poloxamer 407 rat model of HL. The HL state caused unexpected changes in the distribution of HF enantiomers. In contrast to plasma, concentrations of desbutyl-HF (DHF) were much higher in highly perfused tissues. Following in vitro incubation of racemic HF and DHF, HF and DHF enantiomers shifted from the lipoprotein deficient fraction to triglyceride-rich fractions in HL plasma. No significant differences were noted in HF metabolism between NL and HL liver microsomes. It appears that both reduced plasma unbound fraction and lipoprotein associated directed uptake of lipoprotein-bound drug by tissues play roles in enantiomer biodistribution. In everted gut metabolism, formation of DHF enantiomers was inversely proportional to bile concentration whereas addition of lipids in the presence of bile caused a significant decrease in DHF:HF ratio of (-)-enantiomers. Pre-treatment of rats with peanut oil had no significant effect on DHF formation in the incubated sacs or microsomal preparations, nor did it affect the expression of intestinal CYP450. The above results were consistent with previous in vivo evaluations showing that the DHF to HF ratio was decreased by the ingestion of a high fat meal. In the ECG study, HL by itself had no effect on the ECG. In HL rats, plasma but not heart concentrations of the HF enantiomers were significantly higher compared to NL rats. DHF did not impart significant ECG prolonging effects after HF administration. The unbound fraction of HF was the controlling factor for drug uptake by the heart. Despite the lack of difference in HF heart concentrations, the QT and QTc intervals were significantly higher in HL compared to NL rats, suggesting a greater vulnerability towards HF induced QT interval prolongation in the HL state. The HL serum resulted in decreased metabolism of HF enantiomers in the isolated primary rat hepatocytes. Studies with LLC PK1 and NRK 52E cells showed that HF is not a substrate of P-glycoprotein transporters. / Pharmaceutical Sciences

Bone formation and remodeling require continuous generation of osteoprogenitors from bone marrow stromal cells (MSC), which are regulated by local growth factors and hormones with putative roles in mesenchymal proliferation and differentiation. Hepatocyte growth factor (HGF) and its receptor c-Met are widely expressed in MSC and are thought to play a key role in the interactions between cells. 1,25-dihydroxyvitamin D (1,25OHD) is the most active metabolite of vitamin D. 1,25OHD binds to its nuclear/membrane vitamin D receptor (VDR) and generates appropriate biological responses. The purpose of this study was to investigate the regulation of proliferation and differentiation by HGF in human bone marrow-derived stromal cells (hMSC). We examined the impact of HGF on hMSC cell-cycle regulation and the combination effects of HGF and 1,25OHD on hMSC osteogenic differentiation to enhance our knowledge of hMSC regulation. hMSC isolated from bone marrow were plated and grown in DMEM supplemented with 3% FBS incubated at 37C with 5% CO2 in air. HGF treatment of hMSCs reduced the rate of cell proliferation and this result was not due to apoptosis or cell senescence. Real-time RT-PCR and Western blot analysis showed increased gene and protein expression of the cell-cycle inhibitors p53, p21, and p27 after HGF treatment. These results appear to be specific because HGF did not significantly alter the gene expression level of other cell-cycle mediators such as RB, cyclin D1, CDK2, CDK4, or CDK6. Transfection of siRNA specific for cMet, the HGF receptor, eliminated the HGF anti-proliferation effect. cMet siRNA also eliminated the increase in p53, p21, and p27, further supporting a role for these cell-cycle inhibitors in HGF¡¯s regulation of hMSC. These results suggest that treatment of hMSC with HGF slows cell proliferation by increasing the expression of p53, p21, and p27. The reduced rate of cell proliferation did not appear to be due to cell differentiation, because treatment of hMSC with HGF alone did not induce cell differentiation. However, HGF in combination with a known osteogenic differentiation activator, 1,25OHD, significantly increased cell maturation/differentiation compared to 1,25D alone, as indicated by an increase in osteocalcin mRNA (a marker for osteogenic differentiation). Whereas HGF had no effect on 1,25OHD synthesis per se, HGF did induce 1,26OHD receptor (VDR) gene expression. HGF up-regulated the expression of the p63 gene, a member of the p53 family. Knocking down the p63 gene reduced the HGF effect on VDR expression and eliminated the HGF-induced up-regulation of the osteogenic differentiation markers osteopontin (OPN) and bone sialoprotein (BSP). Moreover, the ChIP assay shows that p63 was able to bind to the VDR promoter, possibly explaining the mechanism of p63-mediated VDR up-regulation. These results indicate that HGF can also induce hMSC osteogenic differentiation when combined with 1,25OHD by up-regulating 1,25OHD receptor VDR expression.

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No description available.