标题: Functional Expression of HGF and HGF Receptor/c-met in Adult Human Mesenchymal S [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:38 标题: Functional Expression of HGF and HGF Receptor/c-met in Adult Human Mesenchymal S
a Interdisciplinary Center for Clinical Research on Biomaterials, IZKF BIOMAT, Aachen, Germany;& [/ z' R E; X: @& t+ Y3 V) Z
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b Institute of Pathology, University Hospital, Aachen, Germany 0 e/ p' ]+ b: a& x; {' j' W # t+ V4 [" b# S& z6 UKey Words. HGF ? Mesenchymal stem cells ? Cell migration ? Mobilization% T2 X; _6 f1 A8 l3 Q1 K" N- z
3 U: Z" [! Y9 F0 I" a$ JWilli Jahnen-Dechent, Ph.D., IZKF BIOMAT, University Hospital, Pauwelsstrasse 30, D-52074 Aachen, Germany. Telephone: 49-241-80-80163; Fax: 49-241-80-82573; e-mail: willi.jahnen@rwth-aachen.de - r2 u5 A* M, N* K* A6 ? K* D. v0 ?1 P3 b" [ R
ABSTRACT 9 X- H! w8 S6 |, M( @+ g# C: U( v' {3 R/ P1 y
A classic concept of tissue repair holds that inflammatory cells enter the damaged tissue and signal resident tissue-specific progenitor cells (e.g., parenchymal cells, fibroblasts) for mitosis. Several studies suggest that multipotent (bone marrow) mesenchymal stem cells can also contribute to tissue repair after mobilization, migration, and engraftment of the damaged tissue . In addition, circulating immature cells seem to participate in regeneration of many different tissues . The role of mesenchymal stem cells in tissue remodeling was shown in different in vivo models〞for example, for hepatic regeneration , muscle regeneration , and infarcted myocardium . Not surprisingly mesenchymal and circulating stem cells have attracted increasing attention because they hold great therapeutic potential for endogenous tissue repair and tissue engineering. 9 `1 W4 Y3 ]9 P/ e% ? A9 G: R$ r# A8 p- f+ _ {1 D
Since the original report from Friedenstein et al. , a number of different protocols have been defined to isolate multipotent adult mesenchymal stem cells from bone marrow specimen . Unlike hematopoietic stem cells, human mesenchymal stem cells (hMSC) adhere to cell culture plastic, which is exploited for their isolation . HMSC express CD105 and CD73 but not the lineage-specific surface antigens CD14, CD34, and CD45 . Markers specific for hMSC are not known. Therefore, putative hMSC isolates have to be verified by their capacity to differentiate at least into adipocytes, chondrocytes, and osteoblasts. In addition, bone marrow-derived mesenchymal stem cells can be differentiated in vitro into bone marrow stromal cells and into endothelial, myogenic, hepatic, and neurogenic cells. Cell transplantation studies in human patients and in animals have demonstrated that bone marrow-derived cells can colonize most organs. The colonization was much enhanced by inflammation accompanying, for example, graft rejection or infarction. Hence, successful engraftment of several organs was enhanced by irradiation , chemical injury, and genetic diseases or following infarction . Despite major advances in MSC biology, our knowledge of the signals required for MSC mobilization and migration to the injured tissue site lags behind the extensive experience with hematopoetic stem cells, which are in routine clinical use. Therefore we sought to determine which factors may be responsible for mobilization of hMSC. Hepatocyte growth factor/scatter factor (HGF/SF) is a multipotent growth factor that exerts a mitogenic, motogenic, and morphogenic response on cells expressing c-met, the cellular HGF receptor. HGF/SF is essential in paracrine signaling of mesenchymal and epithelial cells, particularly during embryogenesis, repair, and carcinogenesis . In malignant and transfected cells autocrine stimulation has been described . In pathology, HGF/SF has been shown to induce tumor cell invasion in tissues .- Y Q4 F* ]3 L
* x) K+ T; t0 cStudies on HGF and c-met expression in bone marrow cells were reported by Takai et al. . These authors demonstrated that bone marrow stromal cells constitutively express HGF and promote hematopoiesis. In addition, expression of c-met by stromal cells suggested an autocrine stimulation of stromal cells by HGF. However, it was not determined if HGF and c-met were also expressed by hMSC. This study was undertaken to address this question, including functional aspects of HGF and c-met-like cell migration and proliferation. We demonstrate that HGF and c-met are constitutively expressed by hMSC and that the expression of HGF is downregulated by transforming growth factor-? (TGF-?). Furthermore, HGF exerted a strong chemotactic stimulus on hMSC, which may be further enhanced by autocrine signaling through the HGF c-met pathway. 2 A9 M1 V- O7 Q: x+ {4 `5 i$ r( B% v: g b; D% \$ z4 u
MATERIALS AND METHODS - a9 Y9 D7 k9 ~! U, x 9 v* B& p" s0 c. y4 C& i- qTotal RNA was extracted using guanidinium thiocyanate (RNeasy Kit; Qiagen; Hilden, Germany; http://www.qiagen.com). Reverse transcription was accomplished with 5 μg of total RNA using the "first-strand cDNA synthesis kit" (Amersham Pharmacia Biotech; Buckinghamshire, UK; http://www.amershambiosciences.com) with FPLCpureTM murine reverse transcriptase. PCR was carried out under the following conditions: denaturation at 95～C for 1 min, annealing at 54～C (HGF) or 60～C (c-met) for 1 min, extension at 72～C for 1 min (30 cycles), and a final extension at 72～C for 10 min. PCR amplicon size (266 bp for HGF and 440 bp for c-met) was analyzed by electrophoresis on a 2% agarose gel and visualized with ethidium bromide. The oligonucletoide primer and the GenBank/EMBL identifiers of template sequences were as follows:6 F& R% _( \& z) T2 g7 B
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c-met (NM000245)4 z* ]; p& o1 @
! H6 v4 W' V' H8 D$ [& [# Tforward (nt 1398–1423 ): 5'-AGAAATTCATCA GGCTGTGAAGCGCG-3' ; r1 d) j, ]$ a+ v5 L , d6 {/ E7 ?% E; L ]/ `0 k5 Jreverse (nt 1814–1838 ): 5'-TTCCTCCGATCG CACACATTTGTCG-3'( m8 d. R; K. w: i
" u" R! O6 G: ?! H7 ?' M6 dHGF (XM168542) 1 E3 S' f. l, P: G " G9 }5 r E' r) l) y3 N9 D& U: aforward (nt 548–568): 5'-GGTAAAGGACGCAGC TACAAG-3' 0 e( d. K& {7 v% q ( @9 B( e' j) D8 c( H1 sreverse (nt 794–814): 5'-ATAACTCTCCCCATTGC AGGT-3' 8 u6 M1 {7 B) N" h S ) X2 Y! g& z1 _$ m: [0 hImmunohistochemistry 6 r1 b) ~2 t" {) s7 X T' J4 b" N J" m, L1 i
Subconfluent hMSCs were grown in chamber slides and fixed for 30 min using 0.5% paraformaldehyde (in PBS). After washing with wash buffer (Dulbecco’s phosphate-buffered saline solution A , 0.5% BSA in PBS), unspecific protein-binding capacity was blocked for 15 min using blocking buffer (PBS with 5% BSA). Cells were incubated for 45 min at RT with the first antibody, diluted 1:50 in PBSSA-NP-40 (1:200 dilution of nonidet P-40 in PBSSA), washed three times for 10 min in PBSSA, followed by an incubation with the secondary antibody (1:250 in PBSSA) for 30 min at RT and again three washing steps. FITC-conjugated streptavidin was added (1:250 in PBSSA) for 30 min, protected from light. Finally, cells were washed three times with PBSSA for 10 min and mounted in 4',6'-diamidino-2-phenylindole hydrochloride (DAPI)-containing mounting medium. $ i7 l( f# r5 i# e/ l3 y% T* ^ " t6 y; @2 r+ uPolyclonal rabbit c-met antibody (primary antibody) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA; http://www.scbt.com), and secondary antibody (biotin-conjugated goat anti-rabbit) and FITC-conjugated streptavidin from DAKO. As a negative control, cells were incubated with the first antibody, which had been preincubated overnight at 4～C with a specific blocking peptide (Santa Cruz Biotechnology). ' Q, i$ c A" t: \ , J0 F# b* Q: h% |6 fWestern Blotting , t* V8 w; y8 b9 L: r) Z0 D: w) _; D
Mesenchymal stem cells were lysed with insect cell lysis buffer (PharMingen International; Hamburg, Germany; http://www.pharmingen.com), and protein concentration was determined using the BCA-Kit (Pierce Biotechnology; Rockford, IL; http://www.piercenet.com). Ten μg of protein lysate and protein marker (Perfect Protein AP WB Marker; Novagen; Darmstadt, Germany; http://www.emdbiosciences.com) were separated on 4%-12% SDS gels (NuPAGE; Karlsruhe, Germany) at 70 V for 2.5 hours. Membranes were fixed with methanol, and after electroblotting (60 min, 150 mA) on polyvinylidene fluoride membrane (Bio-Rad; Munich, Germany; http://www.bio-rad.com), the membranes were blocked overnight with low-fat milk at 4～C. Immunostaining was accomplished by incubation with rabbit polyclonal antibody against c-met (1:200, Santa Cruz Biotechnology) for 90 min at room temperature. After 1 hour of incubation with alkaline phosphatase (AP)-conjugated secondary antibody (1:5,000; Roche; Mannheim, Germany; http://www.roche.com) at room temperature, staining was developed using Sigma Fast 5-bromo,4-chloro,3-indoyl phosphate/nitroblue tetrazolium (BCIP/NBT) tablets (Sigma). As a negative control, the first antibody was preincubated with a fivefold excess of blocking peptide in a small volume of PBS at 4～C overnight. - l# O! G9 [. V, {( l3 g: o0 |! X K3 n ^6 H$ q0 P
HGF-ELISA8 a9 \1 N% B/ S4 N( Y
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HGF concentration in hMSC-conditioned media was measured by Quantikine human HGF-ELISA (R&D Systems; Minneapolis, MN; http://www.rndsystems.com), which is based on a sandwich enzyme immunoassay technique with a precoated HGF-specific antibody. To this end, cells were seeded in 96-well plates (20,000 cells/well) and stimulated for 24 hours with TGF-?3 (10, 1, and 0.1 ng/ml), interleukin-1? (IL-1?) (10, 1, and 0.1 ng/ml), and bFGF (10, 1, and 0.1 ng/ml). Unstimulated cells served as a control. Supernatants were harvested and stored at -70～C until they were analyzed. ! Z6 }0 H% S' k7 {: u. S. t# K9 A1 `# \/ |6 J q: Q
Scratch Assay ? hMSC were grown to confluency in a six-well plate (Becton Dickinson). A scratch in the cell layer was made with a pipette tip over the total diameter of 34.5 mm. HGF was added at 0, 25, 50, and 75 ng HGF/ml medium. Closure of this "wound" was documented photographically (Axiovert 25; Zeiss; Cologne, Germany; http://www.zeiss.com) after 24 hours, and cells in four segments of the scratched area, each of 320 μm x 320 μm, were counted. & ?! E' p( l9 f! j8 } $ j: x: s6 c, Q; ^$ l1 yBoyden-Chamber Assay ? For analysis of cell motility, 1 x 105 hMSCs/ml were seeded in the top compartment of a Boyden chamber (NeuroProbe; Gaithersburg, UK; http://www.neuroprobe.com). The bottom compartment contained different HGF concentrations and was separated from the top compartment by a polycarbonate membrane with 8 μm pores (Corning; Düsseldorf, Germany; http://www.corning.com). Cells were allowed to migrate for 16 hours at 37～C in a humidified atmosphere. After removing cells from the upper side of the membrane with cotton swabs, membranes were fixed, stained with hematoxylin (Merck; Darmstadt, Germany; http://www.merck.com), and transferred onto glass slides. Cells on the bottom side of the membrane were counted in five different highpower fields. Each analysis was performed in triplicate. 2 e$ t$ P6 D5 u3 u2 m1 t# z 9 C- B+ x0 m& G7 M! ]Cell Proliferation Assay ? Cells were seeded in 24-well plates (3,000 cells per well) and stimulated for 24 hours with HGF (0, 25, and 50 ng HGF/ml medium) in low serum (2% FCS). The medium containing 20% FCS was used as positive control. Proliferation was measured by detection of ATP content of the cells with a luciferase detection system (ViaLight HS; BioWhittaker; Verviers, Belgium; http://www.biowhittaker.be). This bioluminescent method uses luciferase, which catalyzes the formation of light from ATP and luciferin. The emitted light intensity is linearly related to the ATP concentration . ATP content was measured 1, 2, 3, 4, and 7 days after stimulation. The medium with HGF was renewed after day 4.. m# G; ~4 `( x5 A2 G I
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RESULTS* |- [0 j2 b- g- W/ _+ f& |) X+ L7 z& v
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Adult pluripotent stem cells are thought to reside in many tissues. They can be isolated from skin and adipose tissue , and a baboon study has shown that infused stem cells can colonize a wide range of tissues〞for example, gastrointestinal, kidney, lung, liver, thymus, and skin . However, the amount of stem cells in most tissues is exceedingly low, which leads to the hypothesis that injured tissues produce appropriate cues for engraftment and, furthermore, that the local microenvironment would stimulate the differentiation of engrafted (stem) cells into functional, specialized cells . This begs the question of which signals are necessary and sufficient for stem cell mobilization and recruitment. HGF is well known to augment cell migration, scattering (i.e., "scatter factor"), and proliferation of many different cell types, predominantly of epithelial cells. Furthermore, HGF is considered to be a humoral mediator of organogenesis and morphogenesis of various tissues and organs, as well as regeneration of organs, tumor invasion, and metastasis .% t4 Z% ` {9 D! B9 R+ T
# s% |7 k9 d3 t, w: ~0 f: i+ ]4 z% WIn the bone marrow, HGF is known as an important hematopoietic regulator . Synthesis and release of HGF precursors could be part of an autocrine mechanism. Pre-HGF has to be activated by proteolytic cleavage〞for example, through serine proteases like uPA and tPA, which are also released by hMSC (Neuss et al., unpublished observations). Tissue repair and wound healing are regulated by soluble mediators provided by inflammatory cells. Therefore, we investigated the effect of bFGF, IL-1, and TGF-? on the production of HGF. TGF-?, which is known to be growth inhibitory for epithelial cells, downregulates the production of HGF by hMSC. This effect is not attributed to cell death after TGF-? stimulation (data not shown). In contrast, basic fibroblast growth factor, which stimulates blood vessel formation and angiogenesis , and the proinflammatory cytokine IL-1? have no significant influence on the expression of HGF (determined by ELISA). Nevertheless, bFGF upregulates the production of the pre-HGF-activating serine protease tPA in hMSC (data not shown). Thus, production of mature HGF is regulated by the microenvironment, depending on processes like tissue damage or inflammation. Activated monocytes or macrophages, which are replete at damaged tissue sites, are known to produce HGF/c-met . On a more general note, HGF concentration is increased at sites of tissue damage . After partial hepatectomy in the rat, HGF levels are even measurably elevated in the blood . Taken together, these increases in local and systemic HGF could provide a key chemotactic signal to mobilize and attract hMSC for tissue repair.& v3 E( p& ]8 p# b0 P
( z# W0 r1 l' }1 [+ iHere we identified HGF/SF as a potent regulator of hMSC function, regulating migration and proliferation in vitro. Our study demonstrates the production of HGF and the expression of the HGF receptor c-met by a defined stromal cell population, human mesenchymal stem cells. These cells were shown by FACS analysis to lack any lineage-specific surface marker expression, including CD4, CD14, CD34, and CD117. In addition, the cells were capable of differentiation into adipocytes, chondrocytes, and osteoblasts. Accordingly, these cells represent the bone marrow stromal cell population described by the groups of Pittenger and Haynesworth as MSC. Migration of hMSC was increased by HGF in Boyden chamber assays (chemotactic effect), whereas proliferation of hMSC was negatively influenced. Furthermore, HGF promoted the repopulation of a cell-free "wound" in a cell monolayer wounding model. We attribute this repopulation mainly to the exogenously added HGF, since hMSC did not upregulate HGF production after the cell monolayer was wounded (data not shown).3 p/ q- O% V! l7 {
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Expression of HGF and c-met was also reported by Takai in a bone marrow stromal cell population, but this population was not further characterized. Bone marrow stromal cells are widely used as feeder layers for long-term cultures of hematopoietic stem cells. Due to different isolation and culture procedures, these cells are less homogenous than the MSC used here. It is possible, however, that hMSCs are also contained in bone marrow stromal cells and that, in fact, hMSCs are the source of HGF required for hematopoiesis in the coculture system. This conclusion is supported by the fact that our hMSC produced similar amounts of HGF as did the feeder cells described by Takai et al. and, furthermore, that neither the bone marrow stromal cells nor our hMSC showed increased proliferation in response to HGF. The amount of HGF expressed by hMSC (0.7–2 ng/ml) is higher than the normal HGF serum concentration (0.24–0.33 ng/ml ). As mentioned, HGF concentration can be elevated in the blood after wounding occurs . We suggest that increased systemic HGF may mobilize bone marrow or tissue resident hMSC to colonize damaged target organs. According to our in vitro results, the elevated HGF should also inhibit hMSC proliferation. We speculate that the local environment will ultimately determine hMSC differentiation into mature tissue cells, as is the case in embryonic development. 3 y/ w' }& H6 T( J* o1 ]; ?. \( }0 I4 j: A9 E% J
HGF could possibly help to mobilize autologous hMSC and to direct possible allogeneic hMSC in future stem cell therapy. The role of HGF and its possible role in dysregulation of wound healing (like that described for TGF-? and other growth factors in diabetic mice, for instance) have to be further clarified. The observation that ischemic or traumatic rat brain extracts induced production of HGF in hMSC also supports the idea of HGF as an important factor for tissue repair by (transplanted or autologous) hMSC. The notion that HGF/c-met signaling may be involved in hMSC mobilization and recruitment to damaged tissues is entirely compatible with previous reports that this important regulator of cell motion and differentiation is critically involved in normal development of epithelial tissues , as well as the metastatic spread of tumor cells . ; w/ j8 g ]7 a6 [$ _4 t$ X: L) W) C! d1 @( [; a
ACKNOWLEDGMENT7 K5 N% f- ~( I5 b% f; F
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* These authors contributed equally.% G# }) U9 p: D' k' T3 b' Y; x
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