Expression Profiling and Functional Analysis of Wnt Signaling Mechanisms in Mese



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江边孤钓

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发表于 2009-3-5 10:37 |显示全部帖子

a Biomedical Tissue Research, Department of Biology, University of York, Heslington, York, United Kingdom;

b Smith and Nephew Research Centre, York Science Park, Heslington, York, United Kingdom

Key Words. Mesenchymal stem cell ? Wnt ? ?-catenin ? Osteoblast ? Signaling mechanisms ? Expression profiling! |. X! E0 c. ~+ _

Correspondence: Paul G. Genever, Ph.D., Biomedical Tissue Research, Department of Biology, P.O. Box 373, University of York, York, Y010 5YW, U.K. Telephone: 01904-328649; Fax: 01904-328659; e-mail: pg5@york.ac.uk% V. @5 \6 P$ Z& r5 {% ]% P

ABSTRACT. m% P9 Q' [4 ^& F

Mesenchymal stem cells (MSCs) have great therapeutic potential, with the capacity to influence diverse medical applications, such as tissue engineering and gene therapy. MSCs self-renew and differentiate into a range of mesenchymal tissues, including bone, adipose, cartilage, muscle, marrow stroma, tendon, and ligament, both in vivo and in vitro, under appropriate culture conditions, via a series of increasingly differentiated precursor cells .

Mesenchymal stem cells were first identified in human bone marrow as cells that were able to self-proliferate and also differentiate into connective tissues, such as bone and cartilage . Many groups have since isolated similar cells from bone marrow and expanded colonies in culture, before inducing differentiation into various mesenchymal lineages . These cells, capable of extensive self-proliferation and also multilineage differentiation, have subsequently been assigned several terms, such as mesenchymal progenitor cells, marrow stromal cells, and mesenchymal stem cells. In this rapidly growing area of research, uniformity with regard to cell type classification is currently lacking, and no definitive marker of MSCs is currently available. However, several monoclonal antibodies have been raised against MSCs , providing a panel of markers for characterization of these cells, including SH-2 (CD105, endoglin ), SH-3 and SH-4 (both CD73, ecto-5'-nucleotidase ), SB-10 (CD166,ALCAM ), and, in addition, the cell-surface antigens CD29 (?1-integrin), CD44 (H-CAM), CD90 (Thy-1), and STRO-1 . MSCs are also characterized by their negativity of the hematopoietic markers CD34 and CD45 and have been found to be human leukocyte antigen (HLA) class I positive and HLA class II negative . Here, consistent with previously published research and general consensus in the field, we define MSCs as a discrete population of cells within the bone marrow stromal cell (BMSC) compartment that self-proliferate extensively, express the MSC antigens described above, and exhibit multilineage potential.: J$ Y1 f5 d5 u
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It is clearly desirable to determine the signaling mechanisms involved in controlling MSC activity in order to identify novel therapeutic targets. Previously published work has identified intracellular signaling mechanisms involved in osteogenic and adipogenic specification in MSC cultures , but relatively little is known about the regulatory inputs that enable the MSCs to maintain their undifferentiated phenotype or those that regulate its differentiation into specialized cell types. Recent expression profiling studies have identified key developmental signaling molecules, including Wnts, in pluripotent embryonic stem cell populations, suggesting that these fundamental cell–cell communication pathways may be instrumental in controlling "stemness" . Here we have identified the expression profile of Wnt signaling molecules in MSCs, providing evidence for their involvement in both the maintenance and differentiation of adult mesenchymal progenitor cells.3 P; K( b0 s! D& _, p

Wnt proteins constitute one of the most important families of signaling molecules in development, and these proteins also have vital roles in adult tissues〞for example, in the regulation of cell proliferation and motility, generation of cell polarity, and specification of cell fate . Recently, Wnt signaling has been implicated in the control of differentiation of hematopoietic stem cells  and stem cells in skin , as well as in regulating myogenesis , chondrogenesis , and adipogenesis . A role for Wnt signaling in osteoblast differentiation has also been suggested, predominantly through studies of the Wnt coreceptor, low-density lipoprotein receptor–related protein–5 (LRP-5). Disruptive mutations in this receptor are associated with decreased bone mass, as a consequence of reduced osteoblast proliferation , whereas high bone-mass phenotypes and increased osteoblast proliferation are associated with activating mutations in LRP-5 . Recently, BMP-2 has also been shown to induce alkaline phosphatase and mineralization via Wnt signaling in mesenchymal cell lines .3 q2 [8 S$ T& F# j, D

Wnts are highly conserved, cysteine-rich secreted ligands, and so far 19 have been identified in humans. Wnt signaling can stimulate at least four different signaling pathways, the best characterized being the canonical pathway, which regulates ?-catenin stability, leading to downstream transcription of target genes (Fig. 1; for review, see ). In the absence of a Wnt signal, ?-catenin is phosphorylated by glycogen synthase kinase–3?(GSK-3?), in association with axin and adenomatous polyposis coli (APC), which targets ?-catenin for ubiquitinylation and subsequent degradation by proteasomes. However, when Wnt ligands bind to Frizzled (Fz) receptors, as well as coreceptors LRP-5 and LRP-6, the cytoplasmic protein Disheveled (Dvl) is activated. Phosphorylation of ?-catenin by GSK-3?is inhibited by Dvl, causing ?-catenin stabilization and accumulation, before translocation to the nucleus, where it binds with members of the T-cell factor (TCF) and lymphoid enhancer factor (LEF) transcription factor family, to induce expression of target genes. Secreted Fz-related peptides (sFRPs) have recently been identified as negative regulators of Wnt signaling (reviewed in ). These are structurally very similar to Fz and are thought to compete with Fz for Wnt ligand binding. Dickkopf-1 (Dkk-1) has also been shown to inhibit Wnt signaling  via its association with LRP-5 and -6. Kremen is the Dkk-1 receptor, which functionally cooperates with Dkk-1 to inhibit Wnt signaling . The first report of a successfully isolated Wnt protein was published only last year , as until then bioactivity was often lost in the purification process. However, lithium ion (Li ) application is often used to mimic canonical Wnt signaling, as it inhibits GSK-3?, therefore stimulating downstream components of the Wnt signaling pathway . It is also possible to stimulate Wnt signaling in vitro using conditioned medium collected from a Wnt3a-overexpressing cell line. Here we provide evidence to suggest a consistent Wnt/Fz expression profile across several undifferentiated MSC populations, which appears to support functional, endogenous canonical Wnt signaling in these cells.
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Figure 1. The canonical Wnt signaling pathway. (A): Unstimulated. In the absence of Wnt ligand, ?-catenin is associated with glycogen synthase kinase–3?(GSK-3?), axin, and adenomatous polyposis coli (APC). Following phosphorylation of ?-catenin by GSK-3?, ?-catenin is marked for ubiquitinylation and then degraded by proteasomes. (B): Stimulated. Wnt ligand binding to Fz receptor and LRP coreceptor activates Dvl, which inhibits GSK-3?. ?-catenin accumulates and associates with T-cell factor and lymphoid enhancer factors (TCF/LEF) in the nucleus to regulate target gene transcription. Secreted Frizzled-related peptides (sFRPs) compete with Fz for Wnt ligand binding, thereby regulating signaling levels. Li  inhibits GSK-3?and therefore can be used to mimic canonical Wnt signaling. Abbreviations: Dvl, Disheveled; Fz, Frizzled; LRP, lipoprotein receptor–related protein.+ f( Z. G3 n# N, x- e' V

MATERIALS AND METHODS0 Q6 A/ X( _$ Z( ]+ p$ _
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Characterization of MSCs from BMSCs: n1 g  A' o5 J% [, }3 E
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A series of analyses were performed to confirm that cells isolated from the human bone marrow exhibited MSC characteristics. Primary adherent human BMSCs from six donors were cultured in control medium, and a cell sample was analyzed for expression of MSC markers using flow cytometry at each passage (Fig. 2A). The percentage of cells expressing the MSC markers CD29, CD44, CD73, CD90, CD166, and HLA class I was increased as the cells were cultured from passage 0 to passage 6. Cells were also found to be negative for the hematopoietic markers CD34 and CD45 and HLA-DR antigen. When cultured in appropriate differentiation conditions, these cells could also be induced to undergo osteogenic, adipogenic, and chondrogenic differentiation (Fig. 2B). These cells were designated MSCs.# v# u9 M8 x  d
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Figure 2. Characterization of primary human mesenchymal stem cells (MSCs). (A): Representative flow cytometric analyses of expression of the MSC markers CD29, CD44, CD73, CD90, and CD166; hematopoietic markers CD34 and CD45; and HLA Class 1 and HLA DR antigens by the human bone marrow stromal cell population at passages 0 and 6. (B): Passage 3 MSCs grown in osteogenic medium for 12 days, then stained for alkaline phosphatase activity (ALP) and von Kossa (vK) to identify mineral as black deposits (left), or adipogenic medium for 12 days, before oil red O and hematoxylin staining (Oil R, middle), or chondrogenic medium in micromass cultures for 15 days followed by alcian blue staining, which identified a dense, glycosaminoglycan-rich matrix (Alc B, right).- I2 C) n# b: t5 ~

Expression of Components of the Wnt Signaling Pathway by MSCs and Mesenchymal Cell Lines0 v2 q6 S9 |) `9 G

RT-PCR analysis was used to determine which Wnt-related genes were expressed by primary MSCs, as well as two cell lines commonly used to model MSCs: MC3T3-E1 cells and C2C12 cells. As Wnt signaling is of major importance during development, cDNA from whole mouse embryos was used as a positive control for the Wnt primers when appropriate (Fig. 3). cDNAs from MSCs obtained from femoral heads from three different donors and a commercial source, at different passages, were analyzed, and expression patterns were found to be strikingly similar across all cell populations. Wnt2, 4, 5a, 11, and 16 expression was identified, along with Fz2, 3, 4, 5, and 6, as well as sFRP2, 3, and 4. Other signaling molecules〞LRP-5, Dkk-1, kremen-1, Dvl-1, Dvl-2, Dvl-3, GSK-3?, APC, ?-catenin, TCF1, and TCF4〞were also shown to be expressed by these cells (Fig. 3). Expression of Wnt1, 2b, 3, 3a, 6, 7a, 7b, 8a, 10b, and 14; Fz1, 7, 8, and 10; and LRP-6 was not detected in MSCs using these techniques (data not shown). MC3T3-E1 cells and C2C12 cells were both shown to express Wnt4, 5, 6, 10a, 11, and 14 and Fz1, 4, and 6, with C2C12s also expressing Wnt2, 7b, and 16 and Fz3.
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Figure 3. RT polymerase chain reaction analyses to identify expression of components of the Wnt signaling pathway in primary mesenchymal stem cells (MSCs) from three different donors and at different times in culture. MC3T3-E1 cells and C2C12 cells were also analyzed as these cell lines are often used to model MSCs. Whole mouse embryos, 8 and 10 days after fertilization, were used as positive controls when appropriate (see Table 1). Control reactions (no RT) were performed in parallel using samples generated in the absence of reverse transcriptase. Representative images from one donor are shown; see Table 1 for details of other donors, different times in culture, and MC3T3-E1, C2C12, and mouse embryo cells. Primer sequences are also listed in Table 1. Abbreviations: C.A., commercially available MSCs (Cambrex Bio Science, Walkersville, MD); ND, not determined; no RT, no reverse transcriptase controls; expression detected; –, no expression detected.
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Functional Canonical Wnt Signaling in MSCs
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Having determined expression of many components of the Wnt pathway by MSCs, canonical Wnt signaling was then mimicked in these cells using conditioned medium from a Wnt3a-overexpressing cell line or Li  ions to inhibit GSK-3?and induce ?-catenin stabilization. MSCs were incubated in 20 mM LiCl or 50% Wnt3a conditioned medium, and control cells were treated with 20 mM NaCl, vehicle, or 50% L-cell conditioned medium, for 0–24 hours, before immunofluorescent localization of ?-catenin with DAPI nuclear staining (Fig. 4). In vehicle-treated, NaCl-treated, and L-cell conditioned medium–treated controls, ?-catenin was predominantly immunolocalized at the cell periphery. However, weak ?-catenin immunoreactivity was identified in some nuclei (Fig. 4A). In comparison, nuclear accumulation of ?-catenin immunostaining was markedly increased in MSCs treated with Li  and Wnt3a conditioned medium (Fig. 4A). These observations were quantified, and the percentage cells with nuclear ?-catenin staining increased approximately threefold after 24 hours incubation with 20 mM LiCl or Wnt3a, compared with controls (Fig. 4B). Levels of phosphorylated ?-catenin were increased in control cells following incubation with calyculin A, a serine/threonine phosphatase inhibitor that prevents dephosphorylation of ?-catenin, once it has been phosphorylated by GSK-3?(Fig. 4C). However, after 1 hour treatment with calyculin A, levels of phosphorylated ?-catenin were decreased in MSCs incubated with LiCl, compared with the NaCl- and vehicle-treated controls.
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Figure 4. Effects of Wnt3a and Li  ion exposure on ?-catenin phosphorylation and nuclear translocation in mesenchymal stem cells (MSCs). (A): Immunolocalization of fluorescein isothiocyanate–labeled ?-catenin in MSCs by confocal microscopy. Cells were treated for 24 hours with 50% Wnt3a conditioned medium or 20 mM LiCl, using 50% L-cell conditioned medium, 20 mM NaCl, or vehicle (dH2O) as controls. Arrows indicate nuclei, with open arrows identifying weak ?-catenin immunoreactivity in controls, compared with intense immunoreactivity in Wnt3a- and Li -treated cells (filled arrows). Bar = 5 μm. (B): MSCs were treated with 20 mM LiCl or 50% Wnt3a conditioned medium, or 20 mM NaCl or 50% L-cell conditioned medium as controls, for 4 or 24 hours, before immunolocalization of ?-catenin and 4,6-diamido-2-phenylindole nuclear staining. The percentage of cells with nuclear ?-catenin staining was then calculated for each treatment, and the results obtained after Li  or Wnt3a exposure were normalized against the NaCl or L-cell controls, respectively. After 24 hours of LiCl and Wnt3a treatment, the percentage of cells with nuclear ?-catenin increased significantly, compared with the controls. Mean values shown ± standard deviation (n = 3); *p 4 h& p5 T( H- ]+ k2 a

DISCUSSION

Our evidence suggests that an endogenous level of Wnt signalling is required for the maintenance and functionality of MSCs. These data indicate that further studies concerning the role of specific Wnt and Fz pairings in the MSC will greatly enhance our current understanding of the signaling mechanisms involved in both stem cell maintenance and mesenchymal differentiation.: g! X7 }' B9 [1 E" R* W

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