TLR4 Activation Promotes Bone Marrow MSC Osteogenic Differentiation And Maturation Via Wnt3a And Wnt5a Signaling

Background and ObjectiveAdult bone marrow mesenchymal stem/stromal cells(MSCs) are multipotential stem cells, and have the ability to differentiate into osteoblasts, chondrocytes, tenocytes, neurons, adipocytes, and skeletal myocytes. Due to the multipotent of MSCs, they have become a therapeutic option for several pathologies including osteogenesis imperfecta, myocardial infarction, and wound healing. MSCs play an important role in bone remodeling because they can be induced to differentiate into osteoblasts. Published data have revealed that bone marrow derived MSCs can repair bone defects in animal models. In addition to their multipotential plasticity, MSCs play a critical role in regulating immune responses in a manner that depends on their state of activation. It is also an important cell to form the critical microenvironment for bone regeneration after injury. Therefore, it is important to reveal the molecular mechanisms that regulate the MSCs function including survival, proliferation, differentiation and cytokine secretion.Toll-like receptor 4(TLR4) are the best studied immune sensors of invading microbes. It is broadly distributed on cells throughout the immune system. Activation of TLR4 is essential for inducing the immune responses, and enhances adaptive immunity against pathogens. It has been revealed that MSCs derived from adult bone marrow also express functional TLR4. Activation of TLR4 signaling in MSCs may influence their survival, differentiation, proliferation, migration and pro-inflammatory cytokine secretion. TLR4 recognizes lipopolysaccharides(LPS) from gram-negative bacteria. It has been proved that LPS can protect MSCs from oxidative stress-induced apoptosis and enhance proliferation of MSCs via TLR4 and PI3K/Akt signaling. LPS was also found to promote the osteogenic differentiation in adult human MSCs. However, due to conflicting reports on various effects of TLR4 ligands on MSCs, further studies are still needed to explore the cellular biological changes of MSCs after TLR4 activation in different models. Importantly, the molecular mechanisms by which TLR4 regulating MSCs proliferation and differentiation are largely unknown.Wingless proteins(Wnt) are a family of cysteine-rich glycoproteins that regulate embryonic development, cell proliferation, migration, differentiation, and death. Recently, Wnt signaling has been revealed to function in controlling the cell fate specification and differentiation of MSCs. Previous researches have shown that Wnt signaling has the capacity to regulate the proliferation and migration of MSCs. Moreover, Wnt signaling has also been found involved in the osteogenic and adipogenic differentiation process in human MSCs. Till now, exist evidence have identified that Wnt3 a, Wnt5 a, Wnt6, Wnt10 a, and Wnt10 b signaling are all involved in controlling the stem cell properties of MSCs. Wnt5 a has been shown to be involved in the induction of MSCs osteogenesis and suppression of adipogenesis. Wnt3 a promotes proliferation and suppresses osteogenic differentiation of adult human MSCs. Wnt10 b has been proposed to influence the decision of MSCs to give rise to either an adipocyte or an osteoblast. Knockdown of endogenous Wnt6 is associated with increasing preadipocyte differentiation and impaired osteoblastogenesis. However, little is known about the interactions between TLR4 and Wnt signaling and their function in regulating MSCs proliferation and differentiation.In the present work, we used TLR4 ligand LPS to activate TLR4 in bone marrow MSCs. We studied the effects of TLR4 activation on the survival, proliferation, osteogenic differentiation, and cytokine production of bone marrow originated MSCs in vitro. In addition, we examined the involvement of Wnt signaling in this process, and the exact role of Wnt3 a and Wnt5 a in TLR4-induced MSCs proliferation and osteogenic differentiation.Methods1. Bone marrow cells were obtained from femur and tibia of 6- to 8-week-old male wild mice and TLR4-/- mice. The cells were cultured in vitro to bulid wild MSCs model and TLR4-/- MSCs model. The MSCs phenotype was defined using flow cytometric analysis with the following monoclonal antibodies:CD29, CD44, CD31 and CD45. Genomic DNAs were analyzed by PCR and electrophoresis to idendity whether TLR4 gene was knocked out in TLR4-/- MSCs model.2. Dose-dependent effects of LPS on the proliferation of MSCs was examined using CCK-8 kit assay. The effects of 1000 ng/ml LPS on the survival of MSCs were detected using flow cytometric analysis with Annexin V-FITC and PI staining. Time-dependent effects of LPS on the proliferation of MSCs was examined using CCK-8 kit assay. The effects of LPS on the proliferation of MSCs were confirmed by Ed U incorporation analysis.3. After MSCs were induced for osteogenic differentiation in the presence of 1000 ng/ml LPS for 3, 5, or 7 days, the ALP activity in LPS-treated differentiated cells was detected using an ALP kit, to assess osteogenic differentiation of MSCs. After MSCs were induced for osteogenic differentiation in the presence of 1000 ng/ml LPS for 7, 15, 20, or 25 days, the calcium deposit was detected using Alizarin red staining, to assess maturation of MSCs.4. To reveal the effects of LPS on the cytokine production of MSCs, MSCs were treated with 1000 ng/ml LPS for 3 day. The m RNA production of IL-6, IL-1β, and TNF-α was detected by Real-time PCR, and the concentration of IL-6, IL-1β, and TNF-α in the culture medium was detected by ELISA.5. To reveal the effects of LPS on TLR4 expression of MSCs, MSCs were treated with 1000 ng/ml LPS for 3 day. The mRNA expression of TLR4 in MSCs was detected using Real-time PCR, The protein level of TLR4 in membrane was detected using Western blot analysis.6. To reveal the role of TLR4 deletion on LPS-induced proliferation of MSCs, wild type MSCs and TLR4-/- MSCs were treated with 1000 ng/ml LPS for 6 days. Cell proliferation was examined by CCK-8 kit assay and Ed U incorporation analysis, respectively.7. To reveal the role of TLR4 deletion on LPS-induced osteogenic differentiation and maturation of MSCs, wild type MSCs and TLR4-/- MSCs were differentiated with 1000 ng/ml LPS for 3, 5, or 7 days, the ALP activity was detected by ALP kit. Wild type MSCs and TLR4-/- MSCs were differentiated with 1000 ng/ml LPS for 7, 15, or 25 days, the calcium deposit was detected by alizarin red staining.8. To reveal the role of TLR4 deletion on LPS-induced cytokines production of MSCs, wild type MSCs and TLR4-/- MSCs were treated by 1000 ng/ml LPS for 3 days, the m RNA ecpression of IL-1β and IL-6 were detected by Real-time PCR, respectively.9. MSCs were treated with LPS for 3 days, the mRNA expression of Wnt3 a, Wnt5 a, Wnt6, Wnt10 a, and Wnt10 b was detected by Real-time PCR, to screen the wnts which were changed in expression level.10. To reveal the effects of LPS on the Wnt3 a and Wnt5 a production of MSCs, MSCs were treated with LPS for 1, 2, or 3 days, the m RNA expression of Wnt3 a and Wnt5 a were detected by Real-time PCR, respectively, and the protein level of Wnt3 a and Wnt5 a in the culture medium were detected by ELISA, respectively.11. To reveal the role of TLR4 deletion on LPS-induced Wnt3 a and Wnt5 a production of MSCs, wild type MSCs and TLR4-/- MSCs were treated with 1000 ng/ml LPS for 1, 2, or 3 days, respectively. The mRNA expression of Wnt3 a and Wnt5 a were detected by Real-time PCR, and the protein expression of Wnt3 a and Wnt5 a were detected by ELISA.12. Based on RNA interference technique, MSCs were transfected with Wnt3 a and Wnt5 a si RNA respectively. The mRNA expression of Wnt3 a and Wnt5 a was detected by Real-time PCR 2 and 4 days after transfection.13. To reveal the role of Wnt3 a and Wnt5 a on LPS-induced cytokines production of MSCs, wild type MSCs were transfected with Wnt3 a or Wnt5 a si RNA, respectively. The MSCs were treated with 1000 ng/ml LPS for 2, or 4 days, the mRNA expression of IL-1β and IL-6 was then detected by Real-time PCR.14. To reveal the role of Wnt3 a and Wnt5 a on LPS-induced proliferation of MSCs, wild type MSCs were transfected with Wnt3 a or Wnt5 a si RNA, respectively. The cells were treated with 1000 ng/ml LPS for 6 days. Cell proliferation was detected by CCK-8 kit assay and Ed U incorporation analysis.15. To reveal the role of Wnt3 a and Wnt5 a on LPS-induced osteogenic differentiation and maturation of MSCs, wild type MSCs were transfected with Wnt3 a or Wnt5 a si RNA, respectively. The cells were differentiated in the presence of 1000 ng/ml LPS, ALP activity in MSC-differentiated cells 7 days after LPS treatment was detected by ALP kit, the calcium deposit in MSC-differentiated cells 15 days after LPS treatment was detected by Alizarin red staining.Results1. Almost all of the wild type and TLR4-/- cells cultured in vitro are CD29 and CD44 positive and are also CD31 and CD45 negative. The models of wild type MSCs and TLR4-/-MSCs in vitro were successfully established, and laid the foundation for following trials.2. No change of cell death was induced by 1000 ng/ml LPS as we detected 6 days after treatment, which indicated that 1000 ng/ml LPS could be set as following experiment concentration.3. The effects of LPS on MSCs proliferation were time-dependent and dose-dependent. LPS remarkably enhanced MSCs proliferation at a concentration of 100 ng/ml, and the effects increased significantly again at a concentration of 1000 ng/ml. LPS treatment significantly promoted MSCs proliferation as detected 2 days after treatment, and the effects increased in a time-dependent manner.4. The ALP activity is enhanced notably at 5 and 7 days after LPS treatment, which indicated that LPS enhanced the osteogenic differentiation of MSCs. From the 15 th day after LPS treatment, the calcium deposit was remarkably increased as compared with control, and this effect increased in a time-dependent manner, which indicated that LPS enhanced the maturation of MSCs.5. The m RNA levels of IL-6 and IL-1β in MSCs were both upregulated after treated with 1000 ng/ml LPS for 3 days. Similar results were found in IL-6 and IL-1β protein expression as detected by ELISA 3 days after LPS treatment. No significant change of TNF-α m RNA and protein level was found after LPS treatment.6. The mRNA and protein levels of TLR4 were both upregulated significantly after treated with 1000 ng/ml LPS for 3 days.7. No difference in the cell proliferation of TLR4-/- MSCs was found after LPS treatment for 6 days. The cell proliferation of TLR4-/- MSCs and wild MSCs without LPS treatment was no different. These result indicated that LPS promotes MSCs proliferation via TLR4 receptor activation. TLR4 knock-out does not inhibit MSCs normal proliferation.8. No change in calcium deposit in the differentiated TLR4-/- cells after LPS treatment was found as detected 7, 15, and 25 days after differentiation. In addition, the ALP activity was not changed after LPS treatment for 3, 5, and 7 days. These result indicated that LPS promotes MSCs osteogenic differentiation and maturation via TLR4 receptor activation.9. The effect of LPS on IL-6 and IL-1β production was abolished by TLR4 deletion, which indicated that LPS promotes cytokines production via TLR4 receptor activation.10. The mRNA levels of Wnt3 a, Wnt5 a, Wnt6, Wnt10 a, and Wnt10 b in MSCs after LPS treatment were detected 3 days. Only the m RNA expression of Wnt3 a and Wnt5 a was significantly increased after TLR4 activation by LPS. Both Wnt3 a and Wnt5 a mRNA level were upregulated as detected in all the 3 days after LPS treatment. Furthermore, the protein concentration of Wnt3 a and Wnt5 a in the culture medium was also increased in a time-dependent manner.11. No change of Wnt3 a and Wnt5 a mRNA expressions was found after LPS treatment in TLR4-/- MSCs, which indicated that LPS promotes Wnt3 a and Wnt5 a expression via TLR4 receptor activation.12. Wnt3 a and Wnt5 a silence has little effect on LPS-induced IL-6 and IL-1β secretion, which indicated that Wnt3 a and Wnt5 a are not involved in TLR4-induced cytokine production.13. Wnt3 a silencing remarkably inhibited the effects of LPS on MSCs proliferation. However, Wnt5 a si RNA treatment has little effect on eliminating the effects of LPS on MSCs proliferation. These results indicated that Wnt3 a is critical for TLR4-induced MSCs proliferation, while Wnt5 a plays little effect in this process.14. Wnt3 a si RNA treatment has little influence on changing the effects of LPS on ALP activity and calcium deposit. Wnt5 a silence significantly antagonized the effects of LPS on increasing ALP activity and calcium deposit. These results suggested that Wnt5 a is involved in TLR4-induced MSCs osteogenic differentiation and maturation, while Wnt3 a plays little effect in this process.Conclusion1. LPS promoted BM-MSCs proliferation, osteogenic differentiation, and cytokine secretion in vitro,while TLR4 deletion these effects of LPS on MSCs were eliminated, which indicated that LPS promotes MSCs proliferation, osteogenic differentiation, and cytokine secretion via TLR4 recptor activation.2. LPS promoted wild MSCs upregulating Wnt3 a and Wnt5 a expression, while TLR4 deletion eliminated these effects, which indicated that LPS upregulates Wnt3 a and Wnt5 a expression via TLR4 recptor activation.TLR4 promotes MSCs proliferation via upregulating Wnt3 a, promotes MSCs osteogenic differentiation and maturation via upregulating Wnt5 a. Cytokine secretion of MSCs via TLR4 recptor activation is irrelevant to Wnt3 a and Wnt5 a. Probably other mechanisms exist in mediating the effects of TLR4-induced cytokine secretion in MSCs.