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Cell Biology International (2011) 35, 961–966 (Printed in Great Britain)
Suppression of the PI3K–Akt pathway is involved in the decreased adhesion and migration of bone marrow-derived mesenchymal stem cells from non-obese diabetic mice
Liren Li*1, Yunfei Xia*1, Zhiwei Wang*, Xiaolei Cao†, Zhanyun Da*, Gengkai Guo*, Jie Qian*, Xia Liu*, Yaping Fan*, Lingyun Sun‡, Aiming Sang*2 and Zhifeng Gu*
*Department of Rheumatology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, Peoples Republic of China, †Department of Pathology, Medical College of Nantong University, Nantong, Jiangsu 226001, Peoples Republic of China, and ‡Department of Rheumatology, Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu 21008, Peoples Republic of China


T1DM (type 1 diabetes mellitus) is an autoimmune disease characterized by T-cell-mediated damage of islet β-cells. The pathology of NOD (non-obese diabetic) mouse involves the insulitis induced by infiltration of T-cells, a similar pathogenic mechanism in T1DM patient. BM-MSCs (bone marrow mesenchymal stem cells) are multipotent progenitor cells that can be isolated from a number of sources. Recent studies have shown that transplantation of MSCs to the NOD mice could prevent the process and have the therapeutic effects on T1DM. In our studies, we have found that migration and adhesion of BM-MSCs from NOD mice were suppressed compared with the BM-MSCs from ICR (imprinting control region) mice, accompanying with the abnormal distribution of FAK (focal adhesion kinase) and F-actin (filamentous actin). Further, we have found that the activation of PI3K (phosphoinositide 3-kinase)–Akt pathway was suppressed in BM-MSCs from NOD mice. When the PI3K–Akt pathway was inhibited by LY294002, the adhesion and migration of BM-MSCs from ICR mice were suppressed as well. These results indicated that the suppression of PI3K–Akt pathway is involved in the decreased adhesion and migration of BM-MSCs from NOD mice.


Key words: adhesion, bone marrow, mesenchymal stem cell, migration, non-obese diabetic mice, type 1 diabetes mellitus

Abbreviations: BM-MSCs, bone marrow mesenchymal stem cells, DMEM, Dulbecco's modified Eagle's medium, F-actin, filamentous actin, FAK, focal adhesion kinase, ICR, imprinting control region, LN, laminin, NOD, non-obese diabetic, PI3K, phosphoinositide 3-kinase, T1DM, type 1 diabetes mellitus

1Liren Li and Yunfei Xia contributed equally to this work.

2To whom correspondence should be addressed (email sangam@ntu.edu.cn).


1. Background

MSCs (mesenchymal stem cells) are multipotent progenitor cells that can be isolated from a number of sources, including BM (bone marrow). MSCs have been noted for their ability to give rise to cells of various lineages, including bone, cartilage and adipose tissues. Besides their developmental plasticity and ability to replace injured tissues, MSCs have also been reported for their profound immunomodulatory capabilities in vivo (Friedenstein et al., 1974; Owen and Friedenstein, 1988; Le Blanc et al., 2003). It has been shown that MSCs, when transplanted systemically, are able to migrate to sites of injury in animals, suggesting that MSCs possess migratory capacity. The properties of MSCs make these cells potentially ideal candidates for tissue engineering. The migratory and adhesion potential of MSCs may play an essential role in tissue repair in vivo. In the injury and recovery process, MSCs could migrate to the injury area and start the differentiation process (Chen et al., 2008). In the pancreas, the MSCs could differentiate into islet cells and pancreatic cells. MSCs have great potential to become a new tool in the list of cellular therapies for T1DM (type 1 diabetes mellitus). T1DM is an autoimmune disease characterized by T-cell-mediated damage of islet β-cells. Patients become dependent on insulin all their lifetime at the onset of T1DM (Magni et al., 2009). NOD (non-obese diabetic) mouse is generated by inbreeding of ICR (imprinting control region) and ICR mice in 1980. Autoimmune diabetes in the NOD mouse resulted from a T-cell-mediated destruction of the insulin-producing β-cells that serve as an animal model for studying human T1DM (Millet et al., 2006). Recent studies have shown that transplantation of BM-MSCs to NOD mice could prevent the process and have therapeutic effects on T1DM (Baertschiger et al., 2009; Madec et al., 2009). The principal mechanism is that BM-MSCs inhibit the autoimmune response of islet β cells (Fiorina et al., 2009) or that MSCs differentiate into islet β cells (Larghero et al., 2009). However, the adhesion and migration capability of NOD mice MSCs cell have not been reported, and the mechanisms underlying the migration of these cells remain unclear.

Interactions between the intracellular components and the ECM (extracellular matrix) regulate many aspects of cell function, including adhesion, migration, cytoskeleton organization and differentiation. These interactions are mediated by cell surface receptors that display a range of binding specificities (Friedrich et al., 2004; Mi et al., 2005). A number of intracellular signalling pathways have been involved in the regulation of adhesive functions, including PKC (protein kinase C), PI3K (phosphoinositide 3-kinase) and the small GTP-binding proteins of the Ras and Rho families (Friedrich et al., 2004; Nobes and Hall, 1999; Zhang et al., 2001). Among the proteins implicated in inside–out signalling, PI3K–Akt has been found in many instances to play a crucial role in modulating cell adhesion, spreading and migration. Compounds that activate PI3K enhance the adhesion of cells to a matrix. Pharmacological inhibitors of PI3K prevent not only focal adhesion formation but also stress fibre formation in several cells plated on LN (laminin) (Friedrich et al., 2004).

In the present study, we have found that the abilities of migration and adhesion in BM-MSCs from NOD mice were suppressed compared with the BM-MSCs from ICR mice, and suppression of PI3K–Akt pathway was involved in this abnormality. These studies revealed new pathophysiological mechanisms of T1DM.

2. Materials and methods

2.1. BM-MSC isolation and identification

Mouse BM-MSCs were isolated and cultured as described previously. The experimentation was carried out according to Nantong University animal experimental ethical guidelines. Cells from BM were plated at a density of 1×106 cells/cm2 in non-coated T-25 cell culture flasks (Beckon Dickinson). Growth medium consisted of DMEM (Dulbecco's modified Eagle's medium) with low glucose (Gibco) and 5% fetal bovine serum (HyClone), supplemented with 10 ng/ml vascular endothelial growth factor (Sigma), 10 ng/ml epidermal growth factor (Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma Chemical) and 2 ml of glutamine (Gibco). Cultures were maintained in a humidified atmosphere with 5% CO2 at 37°C. The medium was replaced, and non-adherent cells were removed after 3 days. The medium was changed twice weekly thereafter. The cell monolayer was formed by homogenous bipolar spindle-like cells with a whirlpool-like array within 2 weeks. Flow cytometric analysis showed that the cells were positive for CD29, CD44, CD105, CD166, but negative for CD14, CD34, CD38, CD45 and HLA-DR (Supplementary Figure S1 at http://www.cellbiolint.org/cbi/035/cbi0350961add.htm). After three passages, cells were used for the following studies.

2.2. Cell migration and adhesion assay

For adhesion assay, 3×104 cells were plated on 96-well plates, which were coated with 20 μg/ml LN (Millipore). After 1 h after plating, the cells were fixed with 4% formaldehyde for 30 min and then stained with 0.25% Crystal Violet in 40% methanol for 3 h. After removing the Crystal Violet solution, the plates were washed, and the stains were released by 2% SDS in PBS. The stain intensity was quantified by spectrophotometry (570 nm) using a plate reader (Multiscan Plus, Labsystem).

For the cell migration assay, 3×105 BM-MSCs were placed on 12-well Transwell assay plates. After 12 h plating, the inside cells were removed, and the filter was fixed with 4% formaldehyde for 30 min before being stained with 0.25% Crystal Violet in 40% methanol for 1 h. Photographs were taken using a monochrome CCD (charge-coupled-device) camera. The migration ability was shown as the ratio of the outside cell number to total cells. Each line had three wells assayed, and experiments were repeated three times (n = 9).

2.3. Western blotting

Equal amounts of protein from cell lysate were separated by 10% SDS/PAGE and transferred to PVDF membranes (Millipore). After being blocked with 5% non-fat died milk powder, membranes were incubated with a rabbit monoclonal antibody against PI3K, p-PI3K (phosphorylated PI3K), Akt, p-Akt (Cellsignal). The antibodies could specifically recognize the Ser473 phosphorylation sites on Akt. Then, the membranes were then washed with PBST (PBS plus 1% Tween 20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG for 1 h. The blots were developed using an enhanced chemiluminescence kit (Pierce).

2.4. Immunofluorescence

BM-MSCs were seeded on glass slides overnight. After being fixed in 4% paraformaldehyde for 15 min, the slides were permeabilized by PBS containing 0.1% Triton X-100, blocked in 1% BSA in PBS and then incubated overnight with primary FAK (focal adhesion kinase) antibodies. The staining of FAK was shown with the FITC-conjugated secondary antibody (Jackson Laboratories). To visualize actin microfilaments, the cells were stained with FITC-conjugated phalloidin (Molecular Probes, Inc.).

2.5. Statistical analysis

All data are expressed as means±S.D. and were analysed statistically using one-way ANOVA (analysis of variance) followed by the Newman–Keuls test with P-values <0.05 considered statistically significant. All statistical analyses were performed using SPSS software.

3. Results

3.1. Cell migration and adhesion were inhibited in BM-MSCs from NOD mice

BM-MSCs of NOD mice and ICR mice were cultured in DMEM medium; after three passages, cells were seeded in Transwell. Cell numbers were counted after 12 h. The number of migrated BM-MSCs from NOD mice was only half of that from ICR mice (Figure 1A). Cell adhesion assay showed that, after 1 h of being seeded on LN, the number of adhered BM-MSCs from NOD mice was about half of that from the ICR mice (Figure 1B). These data indicated that, in the NOD mice, the migration and adhesion ability of BM-MSCs were significantly decreased compared with that in the ICR mice.

3.2. The distribution of F-actin (flamentous actin) and FAK were abnormal in BM-MSCs from NOD mice compared with those from ICR mice

To detect the factors associated with the decreased migration and adhesion of BM-MSCs from NOD mice, we investigated the distribution pattern of FAK and F-actin in these cells. As shown, in the BM-MSCs from ICR mice, there were very distinct focal adhesion contacts and stress fibre formation after 1 h on LN (Figure 2). Compared with the BM-MSCs from ICR mice plated on LN, in nearly 90% cells from NOD mice, the FAK diffusely distributed along the leading edges, but in the cells from ICR mice, the FAK was localized in the perinuclear region and diffusely in the cytosol. In 90% of BM-MSCs from NOD mice, there was very limited stress fibre formation with actin staining less prominent in cortical regions compared with BM-MSCs from ICR mice (Figure 2). At the same time, we have also measured the cell area of the two cells. The surface regions of cells from NOD mice were larger than those from ICR mice (data not shown).

3.3. The activation of PI3K–Akt pathway was suppressed in BM-MSCs from NOD mice

To explore the role of PI3K–Akt pathway in BM-MSC adhesion and migration, we analysed the expression and phosphorylation of PI3K and Akt in these cells. As shown, there was no significant difference in total PI3K or Akt expression in these cells, but the phosphorylation of PI3K and Akt were significantly suppressed in NOD mice BM-MSCs compared with the BM-MSCs from ICR mice (Figure 3).

3.4. The inhibitor of PI3K–Akt mimicked dysfunction in cell migration and adhesion in BM-MSCs from ICR mice

To further analyse the critical role of PI3K–Akt in BM-MSC migration and adhesion, the specific inhibitors LY294002 and wortmannin were used. We found that 20 μM LY294002 and 10 μM wortmannin showed the greatest suppressive ability (Supplementary Figure S2 at http://www.cellbiolint.org/cbi/035/cbi0350961add.htm). Then, we chose the 20 μM LY294002 and 10 μM wortmannin in the following experiment. As shown, cell adhesion was significantly suppressed both in BM-MSCs from NOD and ICR mice, and the number of adhered cells from ICR mice was nearly the same as that from NOD mice when the BM-MSCs from ICR mice were pretreated with LY294002 or wortmannin (Figure 4A). Cell migration and adhesion assay also indicated that LY294002 or wortmannin could significantly inhibit cell migration in cells from ICR and NOD mice (Figures 4A and 4B). Pretreatment with LY294002 or wortmannin has no effect on cell viability in BM-MSCs from ICR and NOD mice (Figure 4C). We also found that, after treatment with LY294002 or wortmannin, the distribution pattern of FAK and F-actin in BM-MSCs from the ICR mice was similar to BM-MSCs from NOD mice (Figure 2), but it showed no difference from the NOD mice (Figure 2).

4. Discussion

T1DM remains one of the most challenging health issues worldwide. Use of lifelong immunosuppressants is limited by lack of efficacy and side effects such as serious morbidity. Alternatively, stem cell therapy, such as use of BM-MSCs, has been reported to be promising in managing T1DM. Chao et al. (2008) successfully differentiated umbilical cord MSCs into mature islet-like cell clusters, and these islet-like cell clusters possess insulin-producing ability in vitro and in vivo. Madec et al. (2009) have found that MSCs could prevent autoimmune β-cell destruction by inducing regulatory T-cells. Recently, Fiorina et al. (2009) found that BM-MSCs from BALB/c mouse could prevent and cure diabetes in NOD mice, whereas BM-MSCs from NOD mouse itself did not have a therapeutic effect. These investigations indicated that MSCs may offer a novel cell-based approach for the prevention and treatment of autoimmune diabetes and that BM-MSCs disorder may play an important role in the development of T1DM. To our knowledge, this is the first study to report the adhesion and migration ability of BM-MSCs from NOD mice.

In the present study, we found that the migration and adhesion ability of BM-MSCs from the NOD mice were significantly decreased compared with those from the ICR mice. The cell skeleton, especially F-actin and focal adhesion, played an essential role in cell migration and adhesion. Cytoskeleton is the intracellular network structure constituted of protein, which contains microtubule, microfilament and intermediate fibre (Olk et al., 2009). The three compositions connect with protein and lipid of the cytoplasmic membrane to be the structural basis of cell movement, cell morphology and transmembrane information exchange (Xu et al., 2009). Cytoskeletal changes in accordance with physiology and pathology condition, assembling and disassembling (Safdar, 2009). Microfilament is the most slender, is mainly composed of actin and exists in the form of G-actin or F-actin (Harris et al., 2009). It plays a part in maintaining cell morphology, tight connection of cells and adhesion of the ectomatrix (Saenz-Morales et al., 2009). The polymerizing/depolymerizing dynamic balance of normal cells is an important regulatory factor of cell movement, adhesion and cell division (Benz et al., 2009). According to the specific fluorescence staining with phalloidin, we found that, in BM-MSCs from NOD mice, F-actin was aggregated and dense on the edge of the cytoplasm, but sparse on the edge of the cell nucleus. It is obviously different from ICR BM-MSCs, in which slender F-actin was found to arrange in parallel, following cell polarity to penetrate the whole cell. It is known that dense F-actin could form crico-fibre, which reduces the motor capacity of cells by restraining the protrusion of parapodium. Taken together, these results demonstrated that the decreased BM-MSCs migration and adhesion might be caused by less focal adhesion and abnormal stress fibre formation.

Akt is phosphorylated by PI3K and thereby linked to migration and adhesion. In glioma and carcinoma cell lines, inhibition of PI3K–Akt by LY294002 could decrease cell migration and induce cell apoptosis (Joy et al., 2003). In this study, we found that the expression of PI3K and Akt showed no difference between these cells, but the phosphorylated PI3K and Akt were significantly decreased in BM-MSCs from NOD mice compared with those from ICR mice. We also found that pretreatment with LY294002 could significantly inhibit cell migration in MSCs from the ICR and NOD mice and had no influence on cell viability. Interestingly, after pretreatment with LY294002, the distribution of FAK and F-actin were reversed in the BM-MSCs from ICR mice. These findings indicated that the PI3K–AKT pathway plays an important role in the migration and adhesion ability of BM-MSCs.

5. Conclusion

In conclusion, we found abnormal migration and adhesion of BM-MSCs from NOD mice. The distribution pattern of F-actin and FAK were also abnormal in these cells. Suppression of PI3K–Akt signalling might account for these abnormalities. Future study to explore the mechanisms underlying migration and adhesion dysfunction in BM-MSCs from NOD mice is warranted, since it may provide a new therapeutic strategy for T1DM treatment.

Author contribution

Aiming Sang and Zhifeng Gu designed the experiments as well as the data analysis. Liren Li and Yunfei Xia conducted the experiments and performed the data analysis. Zhiwei Wang, Xiaolei Cao, Zhanyun Da, Gengkai Guo, Jie Qian, Yaping Fan and Lingyun Sun participated in the experimental design, co-ordination, data analysis and draft of the manuscript.

Funding

This work was supported by the Natural Science Foundation of Jiangsu Colleges and Universities [grant number 09KJB320010] and ‘Top Six Types of Talents’ Financial Assistance of Jiangsu Province Grant [grant number 6].

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Received 28 July 2010/7 March 2011; accepted 30 March 2011

Published as Cell Biology International Immediate Publication 30 March 2011, doi:10.1042/CBI20100544


© The Author(s) Journal compilation © 2011 Portland Press Limited


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)