Our data demonstrate that the addition of 1,25D to mesenchymal multipotent cells in a time and dose dependent manner exerts a potent antiproliferative effect, inducing cell cycle arrest, promoting the accumulation of cells in G0/G1 phase without inducing apoptosis. This antiproliferative effect is characterized by a reduction in the number of cells and an increase in cell size compared to the untreated group. Specifically, we demonstrate for the first time, that supplementation with 1,25D decreases cell proliferation and increases cell size through the activation of Rho and Rhou/Wrcth-1 pathways without inducing apoptosis. In addition, we demonstrate that 1,25D regulates genes involved in cell cycle progression and key pro- and anti-apoptotic pathways as well increases survivin expression a potentially novel 1,25D mediated pathway for regulating tissue injury and fibrosis. These advances in our understanding of key mechanistic pathways regulating antiproliferative and anti-apoptotic effects of 1,25D in a multipotent stem cell model provides an important foundation for future therapeutic interventions such as 1,25D analogs to selected activation of induced pluripotent stem cells in the prevention or treatment of presently considered irreversible hypovitaminosis D related chronic medical conditions such as neoplasias and hyperproliferative disorders including premature organ fibrosis. Although the molecular mechanisms underlying the 1,25D antiproliferative effect have been widely studied, the molecular mechanisms in many relevant models remain poorly characterized. In this manuscript we first focused on unfolding the inhibitory effect of supplementing mesenchymal multipotent cells with 1,25D by different approaches. These multipotent mesenchymal cells upon incubation with AZCT are able to differentiate into multiple phenotypes, such as contractile striated muscle cells, differentiated adipocytes and chondrocytes [16
]. The number of muscle and fat cells that appear in treated cultures depends on the concentration of 5-azacytidine (20μM) and the incubation time. Differentiated cells are not observed until 10-15 days after AZCT treatment, when the cells have passed through several division stages [17
]. In our case, we performed our experiments after 4 days when the cells were still in a pre-differentiated stage, which allowed us to study the effects of 1,25D on cell proliferation when cells were still in a division stage.
We showed that incubation of multipotent cells in a time and dose manner decreases cell proliferation as demonstrated by the decreased expression of PCNA, a protein found in the nucleus that acts as a cofactor of DNA polymerase delta and increases the processivity of leading strand synthesis during DNA replication. The antibody against PCNA recognizes a 36-kd protein that is present during the S phase [36
]; however, in response to DNA damage it is ubiquitinated and is associated in the RAD6-dependent DNA repair pathway, so its detection will mark cell proliferation and DNA repair processes [37
]. The data presented in this manuscript also demonstrate that 1,25D decreases cell number and increases cell size, a phenomenon that can be partially attributed to a reduced expression of Rho and Rhou/Wrch1, but not Wnt-1.
The Rho family of proteins plays a central role in regulating cell shape, polarity and locomotion through their effects on actin polymerization, actomyosin contractility, cell adhesion, and microtubule dynamics [38
]. Rho proteins also promote reorganization of the actin cytoskeleton and regulate cell shape, attachment, and motility. It has been shown that overexpression of this gene is associated with tumor cell proliferation and metastasis. Rhou, also known as Wrch-1 (Wnt-regulated Cdc42 homolog) is an atypical member of the Cdc42 subgroup of Rho GTPases. Rhou mRNA level has been shown by Tao et al. to increase in response to Wnt-1 signaling in Wnt-1 transformed mouse mammary epithelial cells [39
]. Previous work with fibroblasts and epithelial cells in culture has shown that Cdc42 contributes to G1 cell cycle progression by promoting entry into G1 and progression to S phase when expressed in quiescent fibroblasts, whereas inhibition blocks serum-induced G1 progression [40
]. Moreover, a short hairpin RNA-mediated knockdown of Wrch-1 was shown to perturb cystogenesis in 3D culture, suggesting that tight regulation of Wrch-1 activity is necessary for normal epithelial morphogenesis [41
Exploring putative mechanisms posited by Eelen and collaborators [12
], we found the addition of 1,25D to mesenchymal multipotent cells blocks the transition from G1 to S1-phase of the cell cycle accumulating cells in G1 by decreasing the expression of cyclins such as Cyclin A2, B1, B2, D1, D3, E1 and F; as well as cyclin dependent kinases (Cdk) such as Cdk2 and Cdk4. Of particular interest is that in this multipotent cell model, 1,25D G1/S-blocking effect was accompanied by an increased expression of p21, a Cdk-inhibitor. However, 1,25D does not increase the expression of p27, making p21 a primary candidate for the antiproliferative effect in C3H 10T1/2 multipotent cells. It is interesting that the stable expression of p27 upon incubation of cells with 1,25D was accompanied by a decreased expression of the F-box protein Skp2 (S-phase kinase-associated protein 2) which, when expressed, positively regulates the G1-S transition by controlling the stability of several G1 regulators, such as the cell cycle inhibitor p27, down-regulating Skp2 and as a consequence causing sustained stabilization of p27, which is a key elements in the antiproliferative and anticancer actions of vitamin D.
In this present work, we found the decrease in cell proliferation was not accompanied by an increase in cell apoptosis. On the contrary, addition of 1,25D to the cell cultures exerts an anti-apoptotic effect, characterized by increased expression of the anti-apoptotic protein Bcl-2, a gene that has been thought to promote cell survival in cancer patients [42
]. Moreover, 1,25D induced a decreased expression of the executioner pro-apoptotic gene caspase 3. These results are in agreement with Zhang et al [44
] who found that 1,25D inhibits hepatocyte apoptosis in rat allografts by regulating apoptotic-associated genes.
Reaffirming the antiapoptotic effect of 1,25D in our cell model is the fact that the gene expression of Apoptosis inhibitor 5 (Api 5/Aac11) increases when the cells have been incubated with 1,25D. Api56/Aac11 is a critical determinant of dE2F1-induced apoptosis in vivo
and in vitro
. It has been described that this functional interaction occurs in multiple tissues, and it is conserved from flies to humans. Interestingly, Api5/Aac11 acts downstream of E2F and suppresses E2F-dependent apoptosis without generally blocking E2F-dependent transcription. [45
It is interesting to note that the gene expression of the proapoptotic members of the Bcl-2 family such as Bax, Bad, Bak and Bok remains unchanged when the cells have been incubated with 1,25D; reinforcing the antiapoptotic effect of 1,25D in this multipotent cell model.
We also found an increased expression of Survivin, the Baculoviral IAP repeat-containing 5 (Birc5). Survivin contains a baculovirus inhibitor of apoptosis repeat (BIR) a protein domain that is a member of the inhibitor of apoptosis protein (IAP) family. Survivin inhibits apoptosis via its BIR domain, by directly or indirectly interfering with the function of caspases. Survivin is also a chromosomal passenger protein that is required for cell division and is expressed in embryonic tissues as well as in the majority of human cancers, but is not expressed in most normal adult tissues [46
]. Recently, emerging evidence suggest that survivin is involved in tissue injury and in wound healing [47
], which is in agreement with our previous publication regarding the antifibrotic effects of 1,25D in the same multipotent cell system [20
A previous report by Wei and colleagues has identified the pro-apoptotic role of activated transcription factor 5 (ATF5) and identified Cyclin D3 as an ATF5-targeted apoptosis-related gene [49
]. The same article showed that the ectopic expression of ATF5 in HeLa cells markedly increased cisplatin-induced apoptosis and the cleavage of Caspase-3, and induced Cyclin D3 mRNA expression via cooperation with E2F1 transcription factor.
In summary, the addition of 1,25D, the active form of vitamin D, to multipotent mesenchymal cells induced a decrease in cell proliferation, characterized by an increase in cell size, a phenomenon possibly linked to decrease in Rho and Rhou/Wrch-1 expression; it also induced cell cycle arrest, promoting accumulation of cells in G0/G1 phase without inducing apoptosis. The data presented in this manuscript provide a mechanistic explanation for the anti-proliferative and anti-apoptototic properties of 1,25D observed in mesenchymal multipotent cells and provides the mechanistic foundation for not only traditional therapeutic interventions such as 1,25D analogs but select activation of induced pluripotent stem cells in targeted clinical studies.