Hepatocyte Growth Factor Effects on Mesenchymal Stem Cells: Proliferation, Migra - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:14 |只看该作者 |倒序浏览 |打印

作者：Giancarlo Fortea, Marilena Minieria, Paolo Cossaa, Daniele Antenuccia, Marilena Salab, Viola Gnocchib, Roberta Fiaccaventoa, Felicia Carotenutoa, Paolo De Vitoc, Patrizia Morena Baldinic, Maria Pratb, Paolo Di Nardoa作者单位：a Molecular and Cellular Cardiology Lab, Department of Internal Medicine, University of Rome Tor Vergata, Italy.b Department of Medical Sciences, University A. Avogadro of Piemonte Orientale, Novara, Italy.c Department of Biology, University of Rome Tor Vergata, Italy 8 ?/ Z! ~. K/ K- }3 b4 z
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      【摘要】
   Hepatocyte growth factor (HGF), a pleiotropic cytokine of mesenchymal origin promoting migration, proliferation, and survival in a wide spectrum of cells, can also modulate different biological responses in stem cells, but the mechanisms involved are not completely understood so far. In this context, we show that short-term exposure of mesenchymal stem cells (MSCs) to HGF can induce the activation of its cognate Met receptor and the downstream effectors ERK1/2, p38MAPK, and PI3K/Akt, while long-term exposure to HGF resulted in cytoskeletal rearrangement, cell migration, and marked inhibition of proliferation through the arrest in the G1-S checkpoint. When added to MSCs, the K252A tyrosine kinase inhibitor prevented HGF-induced responses. HGF¡¯s effect on MSC proliferation was reversed by p38 inhibitor SB203580, while the effects on cell migration were abrogated by PI3K inhibitor Wortmannin, suggesting that HGF acts through different pathways to determine its complex effects on MSCs. Prolonged treatment with HGF induced the expression of cardiac-specific markers (GATA-4, MEF2C, TEF1, desmin, -MHC, &szlig;-MHC, and nestin) with the concomitant loss of the stem cell markers nucleostemin, c-kit, and CD105.
      【关键词】 Met receptor Mesenchymal stem cells Hepatocyte growth factor p Akt
   INTRODUCTION$ i5 z! A& @7 m3 l

Bone marrow mesenchymal stem cells (MSCs) display a great transdifferentiation potential in adult organisms, being able to differentiate in cell lineages different from those of the original tissue .) X* y5 L2 s% B# G/ W
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Hepatocyte growth factor (HGF) is a pleiotropic cytokine of mesenchymal origin, promoting motility, proliferation, invasion, morphogenesis, and survival of a wide spectrum of cells, namely epithelial and endothelial cells . The latter two couple the Met receptor with the ras-mitogen-activated protein kinase (ras-ERK1/2 MAPK) pathway, while Gab-1 binds PI3-kinase efficiently.5 c  ?8 U. u! r; j: q$ c
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Although biochemical and biological responses induced by HGF on primary cultured cells or established cell lines have been extensively studied, little information is presently available on undifferentiated cells. In human embryonic stem cells, HGF induced differentiation into the three embryonic germ layers  demonstrated that the overexpression of HGF by MSCs engrafted into ischemic myocardium can improve their incorporation in the organ, reducing infarct size, improving heart functions, and inducing angiogenesis.! i+ v1 ^, S3 o0 [, ]

The present study was undertaken to investigate (a) the biochemical pathways involved in HGF activity on mouse MSCs isolated from bone marrow in terms of proliferation and migration, and (b) the possible effects on cell differentiation.# ^. i- j  Q0 c7 m, B) K- R5 e
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MATERIALS AND METHODS

Cell Culture* f  [. X* ^+ M5 d3 F. H
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MSCs were obtained from 6-week-old female C3H/He mice femurs according to Friedenstein¡¯s protocol , 7-4, and Ter-119) and then separated by anti-Biotin microbeads-conjugated secondary antibody. Aliquots of the two separated cell subpopulations (Linpos and Linneg) were then stained with anti-Biotin phycoerythrin-conjugated secondary antibody and analyzed with fluorescence-activated cell sorting (FACS). The Linneg fraction was resuspended in complete Iscove¡¯s modified Dulbecco¡¯s medium (IMDM; Cambrex Bio Science, Verviers, Belgium, http://www.cambrex.com) supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cell concentration was adjusted at 0.5 x 106 per cm2. After 4¨C6 days, an adherent population of MSCs appeared. Medium was replenished, floating cells were removed after 7 and 12 days, and the final adherent cell population was used for the experiments. Serum was omitted from the culture medium in the final passage." }. o8 b1 m. x5 A; Q* |

RNA Extraction, Reverse Transcription, and Semiquantitative and Quantitative Reverse Transcription¨CPolymerase Chain Reaction  P( {, H% m1 g) m5 e1 `
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Total RNA was extracted by Trizol Reagent (GIBCO BRL, Gaithersburg, MD, http://www.gibcobrl.com).* A! _( [2 C* C+ z5 @! m2 X# f6 m
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Reverse transcription (RT) was carried out with 2 µg of RNA for each sample using RT M-MLV (Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com) in the presence of random hexamers. Semiquantitative analysis of RNA expression was carried out by RT¨Cpolymerase chain reaction (PCR) by comparing the control transcript (GAPDH ) and the transcript of interest when their amplification was in the exponential phase. The primers used are reported in Table 1. PCR products were size-fractionated in 2% agarose gel electrophoresis.3 P1 T9 p+ o, S  g% X8 Q0 s
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Table 1. Primers used in the study# Z$ [  p: l' ^+ I/ l, k: t7 Q

For real-time RT-PCR, each reaction was performed in a final volume of 20 microl of Universal Master Mix without Amperase Uracyl N-Glycosilase 2X (UMM no UNG, Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com), murine HGF, Met primers, probe 20X (Mm0115182 ml and Mm00434924 ml assays; Applied Biosystems), and 0.5 microl of template cDNA. As active references, 18S RNA (mammalian 18S PDAR; Applied Biosystems) was used. In each experiment, single samples were amplified three times and each experiment was a triplicate. The system used was the 700 Sequence Detection System (Applied Biosystems). In inhibition experiments, cells were concomitantly incubated with K252a tyrosine kinase inhibitor (EMD Biosciences, Merck KGaA, Darmstadt, Germany, http://www.emdbiosciences.com).# N! j6 W' f4 n* ~

Stimulation, Immunoprecipitation, and Western Blot Analysis3 j7 B& H( a% H  O3 U

Quiescent MSCs were incubated for 15 minutes at 37¡ãC in the absence, or presence, of 20 ng/ml of human recombinant HGF (ReliaTech, Braunscwheig, Germany, http://www.reliatech.de), washed twice with cold PBS, and lysed in radio-immunoprecipi-tation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris-HCl ) diluted in Tris-buffered saline¨C5% bovine serum albumin for 2 hours at 22¡ãC.
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MSCs were also treated with 20 ng/ml HGF for 10, 30, and 60 minutes and, after washing with cold PBS, lysed in 100 µl of reducing Laemmli sample buffer; extracts were clarified and protein content quantified by Bradford method (Amresco, Inc., Solon, OH, http://www.amresco-inc.com). Thirty micrograms of the clarified extracts was run in 12.5% SDS-PAGE followed by transfer to a PVDF membrane. Western analysis was carried out using the following primary antibodies: monoclonal antibodies (mAbs) against phosphorylated-ERK1/2 MAPK (Cell Signaling Technology, Inc., Beverly, MA, http://www.cellsignal.com), mAbs against phosphorylated-Akt, phosphorylated-p38, p38 (Sigma-Aldrich, Milan, Italy), polyclonal Abs against Akt1/2, ERK1/2, Met, HGF (Santa Cruz Biotechnology), polyclonal antibodies (pAbs) against p27kip1, p21waf1 (Lab Vision Corporation, Fremont, CA, http://www.labvision.com), and mAb against pRB (BD Biosciences Pharmingen, San Jose, CA, http://www.bdbiosciences.com/pharmingen). After extensive washing, immunocomplexes were detected with horseradish peroxidase¨Cconjugated appropriate secondary antibodies followed by enhanced chemiluminescence reaction (ECLTM; Amersham).

Proliferation Assay

For cell growth assay, 2.5 x 103 cells were seeded in 96-well microplates, grown for 12 hours in 10% FCS and then starved in 2% FCS for 24 hours. Cells were then switched to 2% serum supplemented, or not supplemented, with 20 ng of HGF per ml, or to 10% serum. Fresh medium plus and minus HGF was replenished. Cells were also incubated in 10% FCS, in the presence of 20 ng HGF per ml. Cells were pulsed with 1 µCi per ml -thymidine and incubated for 3 hours. After trypsin treatment, cells were harvested by centrifugation and treated with 5% trichloroacetic acid (TCA) at 4¡ãC for 30 minutes. The TCA-insoluble fraction was resuspended in 0.1% SDS in 200 mM NaOH, and the samples, after addition of 7 ml Optifluor (Packard Instruments, Downers Grove, IL, www.packardbioscience.com), were counted for radioactivity by a liquid scintillation counter (Tricarb 2180 TR, Packard Instruments). As indicated in some experiments, prior to stimulation, cells were preincubated for 1 hour with specific inhibitors. Statistical analysis of the data was carried out using the Student¡¯s t-test.

FACS Analysis7 U8 J5 I% K1 `1 H+ L9 a

To stain isolated nuclei, cells were incubated with 25 µg/ml of propidium iodide (PI; Sigma-Aldrich, Milan, Italy) in a solution containing 2% Triton X-100. In parallel experiments, MSCs were doubly stained with PI and fluorescein isothiocyanate (FITC)¨Clabeled Annexin V (Sigma-Aldrich, Milan, Italy). After 30 minutes of incubation, cells were washed with ice-cold PBS and analyzed in a FACScalibur flow cytometer (BD Biosciences Pharmingen).
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Wound Healing Assay

For wound healing assay, 5 x 105 cells were grown in a 15-mm culture dish and allowed to reach confluence and further incubated in medium containing 2% FCS for 18 hours. The monolayers were then wounded with a plastic pipette as described . After wounding, cells were washed with PBS and incubated for 24 hours in medium containing 2% FCS, with or without HGF (20 ng/ml), in the presence, or absence, of 30 nM of Met tyrosine kinase inhibitor K252a (Calbiochem-Novabiochem Intl.) added immediately before stimulation with HGF, fixed with 11% glutaraldehyde and stained with hematoxylin and eosin. Images of cell samples were taken with a digital camera.

Transwell Migration Assay
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The assay for chemotaxis was performed in Transwell chambers (Corning Costar Italia, Concorezzo, Italy). Briefly, 200 µl of medium containing MSCs was seeded on the upper side of a porous polycarbonate membrane (pore size: 8 µm). Five hundred microliters of medium containing, or not containing, HGF in the presence of 2% FCS was added to the lower compartment. The plates were incubated at 37¡ãC in 5% CO2 for 48 hours. In inhibition experiments, cells were preincubated with K252A inhibitor for 1 hour. At the end of incubation, the cells at the upper side of the filter were mechanically removed. Cells that had migrated to the lower side of the filter were fixed for 30 minutes in 11% glutaraldehyde and stained with hematoxylin and eosin. Five to ten random fields were counted for each filter.+ d' D  B; ]9 Y* H; G7 O$ n
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Immunofluorescence5 O: H! @7 z. J* A. O1 i$ j
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MSCs were seeded on chamber slides (Nalge Nunc International, Rochester, NY, USA, http://www.nalgenunc.com) and treated, or not treated, with 20 ng/ml HGF for 24, 48, 72, and 96 hours. Cells were washed in PBS, fixed in paraformaldehyde 4% in PBS containing CaCl2 for 30 minutes at 4¡ãC and permeabilized with 0.1% Triton X-100. F-actin was labeled with Tetra-rhodamine¨C conjugated Phalloydine in methanol 50% for 30 minutes. Cells were stained with antibodies for c-kit, CD105, GATA-4 (Santa Cruz Biotechnology), nestin (Chemicon International, Temecula, CA, http://www.chemicon.com), and ¨Cmyosin heavy chain (monoclonal antibody MF-20 ). As secondary antibodies, FITC-conjugated mouse anti-goat and goat anti-mouse (Vector Laboratories, Ltd., Peterborough, England, http://www.vectorlabs.com/uk) were used. Nuclei were stained with DAPI (4',6'-diamidino-2-phenylindole). Incubation with FITC-labeled secondary antibody in the absence of specific primary antibody was used to exclude the occurrence of unspecific signals.8 r3 F4 Y; j3 a( C! P$ i" d% h) ^
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RESULTS

Phenotype of the Cells Purified from Bone Marrow
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Cells were negatively selected by magnetic cell sorting on the basis of the expression of Lin surface molecules and positively selected for their ability to adhere to plastic substrate. Before plating on plastic dishes, aliquots of the Linneg and Linpos fractions were analyzed by FACS to assess the preparation quality and the Linneg fraction was found to be 98% pure (data not shown). The cells resulting after plating were c-kitpos, CD105pos, nucleosteminpos, ¨Csmooth muscle actinlow, nestinneg, Sca-1neg, CD34neg, and CD45neg. They did not express any muscle-specific marker.& \2 Q  L+ n  r3 h

MSCs Express Functional HGF and Met Receptor, Both of Which Can Be Upregulated4 Q8 N& c. E/ J. K: z, R

Recently, Neuss and colleagues demonstrated that human MSCs express HGF and its receptor c-Met  were used as positive controls for Met expression.
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Figure 1. Hepatocyte growth factor (HGF) and Met expression in mouse mesenchymal stem cells (MSCs). (A): Reverse transcription¨Cpolymerase chain reaction (RT-PCR) analysis of RNA isolated from quiescent MSCs either untreated (¨C) or treated ( ) with HGF (20 ng/ml) for 48 hours. The Met (top panel) and the HGF (middle panel) major transcripts are detectable in untreated MSCs and upregulated by HGF treatment of 48 hours. RNA from MLP-29 was used as control (C). To verify the quality of RNA, RT-PCR was carried out in parallel with a probe specific for GAPDH (glyceral-dehyde-3-phosphate dehydrogenase) (bottom panel). (B): Quantitative PCR using HGF and Met-specific probes was performed on cDNA obtained from RNA extracted from MSCs treated, or not treated, with HGF in the presence, or absence, of tyrosine kinase inhibitor K252a. (C): Western immunoblotting analysis of Met expression (top panel) and HGF-induced activation (bottom panel) in MSCs. Detergent extracts from MSCs, as well as from cells expressing a physiological level of the Met receptor (MLP-29, positive control), were immunoprecipitated with a mixture of DO-24 and DN-30 Met antibodies, separated by SDS-PAGE, transferred onto membrane, and probed with SP-260 Met antibodies (Met) and antiphosphotyrosine antibodies (P-Met). Similar amounts of proteins were used as detected in Western blot with anti-Met antibodies (top panel). All experiments are representative of three replicates.% a* X) \3 X, W+ n
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HGF Activates ERK1/2, p38 MAP Kinases, and Akt, O) f; w5 l" H" ^3 k

Because HGF activates downstream effectors, such as ERK1/2, p38 MAPKs, and PI3K in several cell types , were performed. Indeed, in these cells, a significant reduction of ERK 1/2 phosphorylation was observed (Taulli et al., personal communication).; p! Z% d% t3 I8 V

Figure 2. Hepatocyte growth factor (HGF) activates ERK1/2, p38 MAPKs, and Akt. (A): Quiescent mesenchymal stem cells were either left unstimulated (¨C) or stimulated with HGF at 20 ng/ml for the indicated times. Total cell lysates were resolved in SDS-PAGE, Western blotted, and immunoprobed with antibodies against the active phosphorylated forms of ERK1/2, p38, and Akt (pERK1/2, pp38, and pAkt) or against the total proteins (ERK1/2, p38, Akt). Protein phosphorylation induced at 10 minutes of stimulation was inhibited if cells were pretreated for 1 hour with specific inhibitors: PD98059 (30 µM PD), SB230580 (30 µM SB), and Wortmannin (100 nM WM), respectively. All experiments are representative of three replicates.5 m1 }3 u8 r4 E6 n/ t, @8 V

HGF Inhibits MSC Proliferation' ^7 j  x4 E. r0 p
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HGF is a pleiotropic cytokine promoting multiple biological effects, namely mitogenesis, motogenesis, survival, and morphogenesis in a cell type¨Cspecific fashion  prompted us to investigate whether this was also the case for MSCs. Indeed, when quiescent MSCs were cultured in medium containing 20 ng/ml of HGF, cell proliferation induced by 10% FCS was nearly abolished (Fig. 3A). FACS analysis performed on stem cells treated, or not treated, with 20 ng/ml HGF for 12, 24, 36, and 48 hours, and stained with PI shows that HGF induces a significant increase in the G0/G1 ratio with respect to control untreated MSCs (Fig. 3B). In a parallel experiment, stem cells were doubly stained with PI and Annexin V to rule out the possibility that HGF treatment could induce cell death by apoptosis or necrosis. Indeed, no increase in the apoptotic or in the necrotic fractions was detectable in MSCs after HGF treatment (data not shown), and, actually, HGF displayed a faint, but reproducible, antiapoptotic activity. We also investigated which signaling pathway was responsible for the HGF-dependent inhibition of FCS-driven cell proliferation, by using specific inhibitors. As shown in Figure 3C, the block in cell proliferation, which was evident within 9 hours, was completely reverted when MSCs were pretreated with the p38 inhibitor SB203580, whereas no effects were observed using PD98059 and Wortmannin, inhibitors of ERK 1/2 MAPK and PI3K, respectively. HGF induced the expression of p21waf1 and p27kip, both of which are recognized as universal cell cycle progression inhibitors. Consistently, the HGF-driven accumulation of the hypophosphorylated forms of Rb, typical of quiescent cells [37), was detected. Finally, HGF treatment induced the expression of bcl-2 and bcl-XL proteins, which is consistent with the antiapoptotic activity displayed by HGF on MSCs (Fig. 3D).; j2 d/ k0 N% Z3 P9 q; Z
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Figure 3. Effects of hepatocyte growth factor (HGF) on mouse mesenchymal stem cell (MSC) proliferation. (A): Cells (2.5 x 103) were seeded in 96-well microplates, grown for 12 hours in 10% fetal calf serum (FCS), and then starved in 2% serum for 24 hours. Cells were switched to 2% serum supplemented with HGF 20 ng/ml (black), or to 10% serum (grey), or to 10% serum containing HGF 20 ng/ml (white), and incubated for the indicated times, with change of medium every second day. Cells were pulsed with 1 µCi/ml -thymidine, incubated for 3 hours, and counted for radioactivity by a liquid scintillation counter (Tricarb 2180 TR). Each value represents the mean of six replicates ¡À SEM. Samples treated with SB203580 were not significantly different from the untreated control samples, indicating that this drug reverted the inhibition exerted by HGF. (D): Western blot analysis of p21waf1, p27kip, pRb (left panel) and of bcl-2 and bcl-XL (right panel) expression in MSCs either untreated or treated with HGF for the indicated times. Bars represent the mean of four independent experiments ¡À SEM. *, p 2 [0 t) S. a5 W. q! @) u
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HGF Activates MSC Migration
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HGF is identical to scatter factor, a molecule that was independently identified for its ability to induce motility in epithelial cells . HGF was also tested in a directional transwell migration assay. In the absence of ligand, MSCs showed a limited ability to cross the filter, whereas, in the presence of HGF (20 ng/ml) in the lower compartment, the number of cells crossing the filter was significantly increased (Fig. 5). This effect was completely inhibited by concomitant incubation with Wortmannin or the tyrosine kinase inhibitor K252a at concentrations mentioned above.. V" |! D6 p# Z& I( ~
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Figure 4. Hepatocyte growth factor (HGF) induces a motogenic response in mouse mesenchymal stem cells (MSCs). A confluent monolayer of MSCs grown in 5% fetal calf serum (FCS) and made quiescent by a 12-hour 2% FCS treatment was ¡®wounded¡¯ with a pipette tip and incubated for 24 hours in the absence (A) or presence of 20 ng/ml HGF (B) or pretreated with PI3K inhibitor Wortmannin (C) or tyrosine kinase inhibitor K252a (D) and then exposed to 20 ng/ml HGF. At the end of the treatment, cells were fixed with 2.5% glutaraldehyde and stained with hematoxi-lin and eosin. MSCs were also plated on cover-slip, and when they reached 60% confluence, they were starved in low serum for 12 hours, untreated (E) or treated with HGF 20 ng/ml (F) for 24 hours, fixed with paraformaldehyde, permeabilized with Triton X-100, and stained for polymerized actin with tetramethylrhodamine isothiocyanate (TRITC)¨Cconjugated Phalloydine. Arrow indicates microspikes protruding from cell surface. Bar = 7.5 µm.3 {; t$ A. d" L( h
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Figure 5. Hepatocyte growth factor (HGF) acts as chemoattractant for mouse mesenchymal stem cells. Cells were untreated (A) or treated with 20 ng/ml HGF (B) or pretreated with tyrosine kinase inhibitor K252a before adding HGF (C) in a transwell directional migration assay. At the end of incubation, the cells at the upper side of the filter were mechanically removed. Cells that had migrated to the lower side of the filter were fixed for 30 minutes in 11% glutaraldehyde and stained with hematoxylin and eosin. Five to ten random fields were counted for each filter (D).
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HGF Prompts MSC Differentiation Toward Cardiac Lineage
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HGF is a potent differentiating factor for human embryonic stem cells and rat bone marrow mesenchymal cells . To test this potential effect on mouse MSCs, cells were cultured for several days in the presence of HGF (20 ng/ml) and the outcoming phenotype was thus examined by determining the expression of some muscle-specific genes using RT-PCR. RNA was isolated from cells treated for different periods of time.4 J- F- O8 ]# q. g% O
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After day 2 of treatment, MSCs lost stem cell markers, like nucleostemin, and started to express mRNAs for the muscle-specific transcription factors MEF2C (myocyte enhancer factor 2C) and TEF1 (transcriptional enhancer factor 1) (Fig. 6). Furthermore, at the same time, they expressed transcripts for desmin, -MHC (¨Cmyosin heavy chain), and ß-MHC, while they did not express other muscular markers, such as MLC 2a (myosin light chain 2 atrial), MLC 2v (myosin light chain 2 ventricular), or ANP (atrial natriuretic peptide) (data not shown). Interestingly, -MHC transcript was detected in a higher amount than ß-MHC transcript. HL-1 cardiac cell line  was used as a positive control for myogenic differentiation of MSCs. Consistently with the onset of a differentiative program, immunofluorescence analysis (Fig. 7) showed that MSCs were no longer reactive for the stem cell markers c-kit (panels A, B) and CD105 (panels C, D) and they showed a positive staining for GATA-4 (panels M, N), a crucial transcription factor operating during the early phases of cardiac development, the intermediate filament protein nestin (panels E, F), and -MHC, a component of the cardiac contractile system (panels G, H).

Figure 6. Hepatocyte growth factor (HGF) induces the expression of myocyte-specific transcription factors and structural genes in mesenchymal stem cells. Cells were untreated (¨C) or treated with 20 ng/ml of HGF ( ) for 48 hours. Total RNA was isolated and after DNase I treatment, reverse transcription¨Cpolymerase chain reaction was performed with primers specific for nucleostemin (NST), myocyte enhancer factor 2C (MEF2C), transcriptional enhancer factor 1 (TEF1), ¨Cmyosin heavy chain (-MHC), ß¨Cmyosin heavy chain (ß-MHC), desmin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as described in Materials and Methods. HL-1 cells were used as positive controls. All experiments are representative of three replicates.
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Figure 7. Hepatocyte growth factor (HGF) induces differentiation of mesenchymal stem cells. Cells were plated on coverslip, and when they reached 60% confluence, they were untreated (A, C, E, G, I) or treated with 20 ng/ml of HGF (B, D, F, H, J) for 96 hours. They were then fixed with paraformaldehyde, permeabilized with Triton X-100, and stained with antibodies for c-kit (A, B), CD105 (TGFßrIII) (C, D), nestin (E, F), MHC (myosin heavy chain) (G, H), and GATA-4 (I, J), followed by the appropriate FITC (fluores-cein isothiocyanate)¨Clabeled secondary antibodies. DAPI (4',6'-diamidino-2-phenylindole) counterstaining was used to visualize nuclei. Bar = 12.5 µm. In control experiments, in which the primary antibody was omitted, no fluorescent signal was detected. All experiments are representative of three independent replicates.( P0 z1 G9 ~& C
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DISCUSSION
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In this study, we investigated the effects of HGF on murine MSCs and found that HGF can induce early biochemical effects, such as receptor tyrosine phosphorylation and upregulation, activation of the major signaling pathways, as well as delayed biological responses, namely block of proliferation, cytoskeletal rearrangement, cell migration, and expression of cardiac-specific markers with concomitant loss of stem cell markers. We also show in some experiments that these effects were dependent on HGF stimulation, because they could be inhibited by the natural alkaloid K252a, which was shown to strongly impair the oncogenic properties of Met . Finally, we present evidence that cell migration is PI3K-dependent, whereas inhibition of cell proliferation is p38-dependent.
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Adult stem cells are attracting increasing attention because of their potential use in both developmental biology and medical applications, such as tissue and organ repair . The fate of these stem cells is determined by microenvironment and growth/differentiation/mobilization factors, which can recruit them in different organs and situations, such as after injury.

HGF is a pleiotropic cytokine displaying mitogenic, motogenic, morphogenetic, and antiapoptotic activities in a cell type¨Cspecific manner . These findings show that HGF can induce biological responses in cells other than epithelial.
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More recently, a few studies have shown that HGF can induce biological responses also in stem cells .9 N$ i; P: A$ l/ ^
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Our results show that mouse MSCs coexpress functional forms of Met receptor as well as of its ligand. Yet, the low levels of HGF found in the culture medium of control samples are not sufficient to activate its own receptor on MSCs; the receptor, however, maintains the biochemical and biological responsiveness to exogenously added ligand. In fact, both the receptor and the ligand can be upregulated upon long-term HGF treatment. Indeed, it was already reported that Met receptor behaves as a delayed early gene, the expression of which in epithelial cells could be upregulated by treatment with serum, phorbol esters, and HGF itself .2 Z! P! C3 b# Q0 c
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In our system, HGF activated cell migration in a PI3K-dependent way, because the effect of wound healing could be blocked by the PI3K pharmacological inhibitor Wortmannin.

Surprisingly, HGF inhibited cell proliferation by blocking cells in the G0-G1 phase. This response was accompanied by the induction of p21waf1 and p27kip proteins, which are known as universal cell cycle progression inhibitors, acting through their binding to cyclins-CDK complexes and PCNA ." I& Z( K1 Q% O+ ^% `- {& a

In our model, upon 48 hours of HGF treatment, MSCs started to express mRNAs for MEF2C and TEF1, two transcription factors, and desmin, which are typically detectable during the first stages of muscle differentiation. Moreover, MSCs expressed transcripts for contractile proteins, such as -MHC and ß-MHC. In this transition toward myogenic differentiation, MSCs lost the expression of markers typically associated with the stem cell phenotype, such as CD105 (TGFßRIII), c-kit, and nucleostemin; remarkably, after 7 days of exposure to HGF, MSCs expressed nestin, a poorly organized form of MHC, and the transcription factor GATA-4, which is crucially involved in early phases of cardiac development. By contrast, these cells failed to express other muscle markers, such as MLCs or ANP, suggesting that further differentiative steps probably require additional factors. Other treatments were also reported to drive bone marrow cells toward the cardiomyocyte phenotype: for example, 5-azacytidine was able to induce morphologic, biochemical, and functional cardiomyocytes , but these cells did not integrate with resident myocytes, again suggesting that additional factors are required for their full functionality.8 }9 n6 p/ @% D% x6 e+ D4 l# M

In conclusion, our findings suggest that HGF could be one of the factors involved in the mobilization and commitment of MSCs toward a cardiomyocyte phenotype even if more detailed experiments will be necessary to clarify whether these processes occur in vivo as well." G: q5 o- i, o6 w" m; ~8 V- d

ACKNOWLEDGMENTS

The authors are grateful to Isabella Screpanti (University of Rome ¡®La Sapienza¡¯), Andrea Graziani (Universit¨¤ del Piemonte Orientale ¡®A. Avogadro¡¯) for helpful discussion throughout these studies and for critically reading the manuscript. The authors thank also Riccardo Taulli (Universit¨¤ di Torino) for kindly providing the lentivirus-siRNA for Met and Syntech srl, Roma, Italy for its technical assistance. This study was supported by grants from Ministero Istruzione, Universit¨¤ e Ricerca (FIRB 2001 and PRIN 2003), ¡®Compagnia di S. Paolo¡¯, Torino, University of Piemonte Orientale ¡®A. Avogadro¡¯, No-vara, and Regione Piemonte (Ricerca Scientifica Applicata 2004-CIPE), Italy., [& N' u# G+ t2 Q& c; L

DISCLOSURES
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The authors indicate no potential conflicts of interest.* A* D& O1 M( X
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