Galectin-3 Enhances Migration of Minature Pig Bone Marrow Mesenchymal Stem Cells Through Inhibition of RhoA-GTP Activity

Bone marrow mesenchymal stem cells (BM-MSCs) are used in tissue engineering because of their migration characters. However, BM-MSCs have limitations in terms of reaching injuries and self-renewal. Therefore, enhancement of BM-MSC migration is important for therapeutic applications. Here, we assessed whether galectin-3 (Gal-3) increases the migration of minature pig BM-MSCs. Gal-3 was knocked down by short hairpin RNA (shRNA) or overexpressed using a lentiviral vector in Wuzhishan minature pig BM-MSCs. Proliferation and migration assays showed that knockdown of Gal-3 impaired BM-MSC proliferation and migration, whereas Gal-3 overexpression promoted these behaviors. RhoA-GTP activity was upregulated in Gal-3 shRNA-transfected BM-MSCs, while Rac-1- and Cdc42-GTP showed no changes. Western blotting indicated downregulation of p-AKT (ser473) and p-Erk1/2 after serum starvation for 12 h in Gal-3-knockdown BM-MSCs. p-AKT (ser473) expression was upregulated after serum starvation for 6 h, and p-Erk1/2 expression was unchanged in Gal-3-overexpressing BM-MSCs. Treatment with C3 transferase or Y27632 enhanced migration, whereas Gal-3 knockdown impaired migration in treated cells. These results demonstrate that Gal-3 may enhance BM-MSC migration, mainly through inhibiting RhoA-GTP activity, increasing p-AKT (ser473) expression, and regulating p-Erk1/2 levels. Our study suggests a novel function of Gal-3 in regulating minature pig BM-MSC migration, which may be beneficial for therapeutic applications.

Scientific RepoRts | 6:26577 | DOI: 10.1038/srep26577 To clarify whether Gal-3 influences minature pig BM-MSC proliferation and migration, we first established stable minature pig BM-MSC lines with Gal-3 knockdown or overexpression. In these cell lines, we examined the underlying mechanism of migration enhancement by detecting changes in RhoA-GTP activity as well as p-AKT (ser473) and p-ERK1/2 levels. Our findings suggest a novel function of Gal-3 in regulating minature pig BM-MSC migration. Gal-3 might be a potential target for clinical applications of BM-MSCs. Our data provide a new perspective for MSC therapeutic strategies for tissue repair and regenerative medicine in the future.

Materials and Methods
Ethics statement. The present study was conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Chinese Academy of Agricultural Sciences (Beijing, China). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Institute of Animal Science, Chinese Academy of Agricultural Sciences.
A lentiviral overexpression vector ( Supplementary Fig. S4) was designed and synthesized based on the miniature pig Gal-3 gene sequence by Shanghai GenePharma Co., Ltd. A negative control was also produced according to the manufacturer's protocol. The virus titer was 1 × 10 9 . Lentivirus-Gal-3 or lentivirus-NC was transduced into BM-MSCs. Stably transfected cell lines were obtained in culture medium containing puromycin (4 μ g/ml). The culture medium was changed every 2 days. Selection was performed for about 1 week.

Statistical analysis.
Results are presented as the mean ± SEM. Statistical analyses were performed by one-way analysis of variance and the Student's t-test. P < 0.05 indicated statistical significance.

Establishment of stable Gal-3-knockdown and -overexpressing BM-MSC lines.
We established stable cell lines of Gal-3-knockdown and -overexpressing minature pig BM-MSCs. The RNA interference vector was transfected into BM-MSCs. Colonies were observed after selection with G418 for about 7 days, and stable cell lines were obtained after selection for about 14 days (Fig. 1A). Similarly, BM-MSCs were infected with the lentivirus. Stable Gal-3-overexpressing cell lines were obtained after selection with puromycin for about 1 week (Fig. 1B). There were high transfection efficiencies and no apparent effects on morphology. Gal-3 shRNA transfection obviously reduced the expression of Gal-3 at both mRNA (1.00 ± 0.04 in shRNA-NC cells and 0.70 ± 0.02 in shRNA-Gal-3 cells, p = 0.0022) and protein levels. Similarly, Gal-3 lentivirus transduction highly increased the expression of Gal-3 at both mRNA (1.00 ± 0.02 in lentivirus-NC cells and 3.53 ± 0.12 in lentivirus-Gal-3 cells, p < 0.0001) and protein levels (0.9396 ± 0.0030 in shRNA-NC cells and 0.4596 ± 0.1543 in shRNA-Gal-3 cells, p < 0.0001; 0.4242 ± 0.0272 in lentivirus-NC cells and 1.157 ± 0.0293 in lentivirus-Gal-3 cells, p < 0.0001, Fig. 1C,D). After successive passages, the expression status of Gal-3 was stable.

Gal-3 enhances proliferation and migration of BM-MSCs in vitro.
The proliferation assay revealed that Gal-3 enhanced the proliferation of BM-MSCs in vitro. We detected the proliferation rate of shRNA-Gal-3 and lentivirus-Gal-3 cell lines, and found that the proliferation rate of shRNA-Gal-3 cells was lower than that of shRNA-NC cells on day 5 (1.15 ± 0.03 in shRNA-NC cells and 0.99 ± 0.03 in shRNA-Gal-3 cells, p = 0.0004) ( Fig. 2A), and the proliferation rate of lentivirus-Gal-3 cells was significantly higher than that of lentivirus-NC cells on day 4 (0.43 ± 0.02 in lentivirus-NC cells and 0.56 ± 0.03 in lentivirus-Gal-3 cells, p = 0.004) (Fig. 2B).

Gal
To further understand the relationship between RhoA-GTP activity and migration, shRNA-Gal-3 and shRNA-NC BM-MSCs were treated with the RhoA inhibitor C3 transferase or Rho kinase inhibitor Y27632. Transwell assay showed enhancement of shRNA-Gal-3 and shRNA-NC BM-MSC migration compared with  (Fig. 3B,C). Therefore, Gal-3 enhanced the migration of BM-MSCs through inhibition of RhoA-GTP activity.

p-Akt (ser473) and p-Erk1/2 are involved in Gal-3-regulated migration of BM-MSCs. To further
understand the underlying mechanism of Gal-3 in BM-MSC migration, we detected the expression of p-Akt (ser473) and p-Erk1/2 in both shRNA-Gal-3 and lentivirus-Gal-3 BM-MSCs by western blotting.
Knockdown of Gal-3 significantly decreased the expression level of p-Akt (ser473) after serum-free starvation for 12 and 24 h. Gal-3 overexpression in BM-MSCs led to an increase in the expression of p-Akt (ser473) in serum-starved cells for 6 and 12 h (Fig. 4). These data indicate that BM-MSC migration stimulated by Gal-3 is likely mediated through increased phosphorylation of AKT (ser473). Next, we found that knockdown of Gal-3 decreased the expression level of p-Erk1/2 in serum-starved BM-MSCs for 12 h. However, no changes were observed in the p-Erk1/2 expression of lentivirus-Gal-3 BM-MSCs, irrespective of serum starvation (Fig. 5). In conclusion, Akt and Erk are involved in Gal-3-regulated migration of BM-MSCs.

Discussion
Gal-3 has multiple properties and biological activities related to many kinds of diseases 28,29 . It is also a regulator of cell migration, and many diseases are related to cell migration 30 , but the exact functions and mechanisms of Gal-3 remain elusive. Enhancement of stem cell migration might contribute to stem cell therapy. Therefore, it would be beneficial for therapeutic applications to understand the function of Gal-3 in regulating BM-MSC migration. Therefore, we established stable Gal-3-knockdown or -overexpressing minature pig BM-MSC lines. Our findings suggest a possible mechanism in which Gal-3 enhances BM-MSC migration through mainly inhibition of RhoA-GTP activity, increasing p-AKT (ser473) expression, and regulating p-Erk1/2 levels (Fig. 6).
Cell proliferation is related to activation of ERK and PI3K/Akt or inhibition of the P38MAPK signaling pathway 21 , which partly supports our data, implying that these signal pathways may be important for minature pig BM-MSC proliferation.
Migration is closely related to various pathological processes involved in vascular and chronic inflammatory diseases including multiple sclerosis and cancer. Polar structure establishment, dynamic processes of actin and microtubule polymerization, regulation of spatial and temporal signal transduction, and other aspects of cell migration have been studied in the past few years 31 . Thus, elucidating cell migration mechanisms is important. In our study, we found that galectin-3 enhances wuzhishan miniature pig BM-MSC proliferation and migration through mainly inhibition of RhoA-GTP activity, increasing p-AKT (ser473) expression, and regulating p-Erk1/2 levels. Rho family proteins, also called Rho GTPases, are guanosine triphosphate (GTP)-binding proteins belonging to the Ras superfamily. The Rho GTPase family plays an important role in regulation of cytoskeleton reorganization 32 as well as many cellular behaviors including cell growth, differentiation, and migration 33,34 . RhoA, Rac-1, and Cdc42 are key regulators in multiple signaling cascades. They mainly regulate morphological changes and the processes of invasion and metastasis in tumor cells 35 . Rac and Cdc42 induce pseudopodia formation, matrix and extracellular matrix degradation 36 , and contribute to cell invasion and metastasis 37 . RhoA is a key member of the Rho GTPase family, which has been identified as an important protein in the regulation of cell migration.  RhoA is mainly involved in stress fiber formation and focal adhesion complex assembly. Although the importance of RhoA has been demonstrated in cell migration, the exact mechanism of RhoA signaling in minature pig BM-MSC migration has not been clarified completely. Our results demonstrated upregulation of RhoA-GTP activity in shRNA-Gal-3 BM-MSCs. After treatment with C3 transferase or Y27632, transwell assays showed enhancement of shRNA-Gal-3 and shRNA-NC BM-MSC migration, and shRNA-Gal-3 BM-MSC migration was lower compared with shRNA-NC BM-MSC migration. Based on these data, it is clear that Gal-3 promotes BM-MSC migration through downregulation of RhoA-GTP activity. We also examined the relationship of Rac1and Cdc42-GTP activities and Gal-3 in cell migration. The results revealed no changes in Gal-3-knockdown or -overexpressing BM-MSCs. Gal-3 enhancement of minature pig BM-MSC migration was not mediated through regulation of Rac1-or Cdc42-GTP activities. Rho regulates several biological behaviors and functions by activating the downstream target molecule Rho kinase. The RhoA/Rho kinase signaling pathway is also associated with various diseases 38,39 . Our data showed upregulation of RhoA-GTP activity in shRNA-Gal-3 BM-MSCs. Therefore, we hypothesized that RhoA inhibition might induce migration of BM-MSCs 40 . We detected cell migration by transwell assays after treatment with RhoA inhibitor C3 transferase or the Rho inhibitor Y27632 that is also a Rho kinase inhibitor. The data confirmed our hypothesis. There was no change in RhoA-GTP activity of lentivirus-Gal-3 BM-MSCs. Therefore, Gal-3 may affect BM-MSC migration to some extent via the RhoA/Rho kinase signaling pathway.
Furthermore, we detected the levels of p-Akt (ser473) and p-Erk1/2. Our data identified a relationship between p-Akt (ser473), p-Erk1/2, and Gal-3 in BM-MSC migration. After serum-free incubation for 12 h, phosphorylation of Akt (ser473) was upregulated in lentivirus-Gal-3 BM-MSCs, and BM-MSC migration increased. These data suggest that BM-MSC migration stimulated by Gal-3 is likely mediated through increased phosphorylation of AKT (ser473). However, when lentivirus-Gal-3 BM-MSCs were incubated in serum-free medium for 24 h, p-Akt(ser473) was hardly expressed, possibly leading to suppression of cell migration. The specific mechanisms will require further study. We also found a decrease in Erk1/2 phosphorylation, along with suppressed migration of shRNA-Gal-3 BM-MSCs that were serum starved for 12 h. However, no change was observed in the expression of p-Erk1/2 in lentivirus-Gal-3 BM-MSCs. Therefore, we propose that Gal-3 may enhance minature pig BM-MSC migration by increasing p-AKT (ser473) expression and regulating p-Erk1/2 levels.
In summary, we showed that Gal-3 plays an important role in promoting minature pig BM-MSC proliferation and migration. Gal-3 may enhance BM-MSC migration through mainly inhibition of RhoA-GTP activity, increasing p-AKT (ser473) expression, and regulating p-Erk1/2 levels. These results may lead to potential applications of Gal-3 in cell therapy. Our findings suggests a novel function of Gal-3 in regulating minature pig BM-MSC migration, and Gal-3 may be a potential target in BM-MSCs for clinical applications.