LIGHT (TNFSF14) Increases the Survival and Proliferation of Human Bone Marrow-Derived Mesenchymal Stem Cells

<div><p>LIGHT (HVEM-L, TNFSF14, or CD258), an entity homologous to <u>l</u>ymphotoxins, with <u>i</u>nducible nature and the ability to compete with herpes simplex virus <u>g</u>lycoprotein D for <u>h</u>erpes virus entry mediator (HVEM)/<u>t</u>umor necrosis factor (TNF)-related 2, is a member of the TNF superfamily. It is expressed as a homotrimer on activated T cells and dendritic cells (DCs), and has three receptors: HVEM, LT-β receptor (LTβR), and decoy receptor 3 (DcR3). So far, three receptors with distinct cellular expression patterns are known to interact with LIGHT. Follicular DCs and stromal cells bind LIGHT through LTβR. We monitored the effects of LIGHT on human bone marrow-derived mesenchymal stem cells (BM-MSCs). At first, we checked the negative and positive differentiation markers of BM-MSCs. And we confirmed the quality of MSCs by staining cells undergoing adipogenesis (Oil Red O staining), chondrogenesis (Alcian blue staining), and osteogenesis (Alizarin red staining). After rhLIGHT treatment, we monitored the count, viability, and proliferation of cells and cell cycle distribution. PDGF and TGFβ production by rhLIGHT was examined by ELISA, and the underlying biological mechanisms were studied by immunoblotting by rhLIGHT treatment. LTβR was constitutively expressed on the surface of human BM-MSCs. Cell number and viability increased after rhLIGHT treatment. BM-MSC proliferation was induced by an increase in the S/G₂/M phase. The expression of not only diverse cyclins such as cyclin B1, D1, D3, and E, but also CDK1 and CDK2, increased, while that of p27 decreased, after rhLIGHT treatment. RhLIGHT-induced PDGF and TGFβ production mediated by STAT3 and Smad3 activation accelerated BM-MSC proliferation. Thus, LIGHT and LTβR interaction increases the survival and proliferation of human BM-MSCs, and therefore, LIGHT might play an important role in stem cell therapy.</p></div

Proposed pathway of LIGHT and LTβR interaction in human BM-MSCs.

<p>LIGHT and LTβR interaction increases the survival and proliferation of human BM-MSCs by activating survival proteins, anti-apoptotic proteins, CDKs, and cyclins. Moreover, LIGHT-induced STAT-3 and smad-3 activation causes PDGF and TGF-β production, and they enhance LIGHT signals in human BM-MSCs. Therefore, LIGHT may play an important role in stem cell therapy, and contribute to MSC modification.</p

Radotinib regulates several signaling pathways.

<p>Kasumi-1 cells were incubated with 5 μM radotinib for 48 h. Cells were harvested, total RNA was isolated and subjected to microarray analysis, as described in the Materials and Methods. (A) The number of genes in categorized pathways affected by radotinib. (B) The percentage of distribution of radotinib-affected genes in each pathway.</p

HPLC analysis and the structure of radotinib.

<p>(A) Radotinib was analyzed by HPLC as described in the Materials and Methods section. (B) The chemical structure of radotinib.</p

RhLIGHT increases the number of human BM-MSCs.

<p>Cells were incubated with 0, 100, and 200 ng/mL rhLIGHT for 72 h. (A) Images of low density (left panel) and high density (right panel) by BSA-control treatment (0.1% BSA-PBS buffer, upper panel) and rhLIGHT treatment (lower panel) in the BM-MSCs. (B) Dose-dependent effect of rhLIGHT on the number of human BM-MSCs at 72 h. (C) Time-dependent effect of rhLIGHT (200 ng/mL) on the number of human BM-MSCs. Data represent the mean ± SEM. Significantly different from the control cells (*); ***, <i>P</i> < 0.001. BSA, bovine serum albumin.</p

Radotinib induces CD11b⁺Annexin V⁺ cells in AML cell lines.

<p>Cells were incubated with various concentrations of radotinib and/or ATRA for 72 h, harvested and immunostained with anti-human antibodies against CD11b and Annexin V, as described in the Methods. (A) NB4. (B) HL60. (C) Kasumi-1. (D) THP-1. (E) The CD11b⁺Annexin V⁺ cells in HL60. (F) Annexin V⁺ in the CD11b⁺ gated cells. (G) Schedule of differentiation induced cell death (plans A, B, C and D). (H) Cell death data by the respective plan. Data represent the mean ± SEM. Statistically significant differences from the DMSO-treated control (*) or ATRA treatment (#) are denoted as follows.*, #: <i>P</i> < 0.05; **, ##: <i>P</i> < 0.01; ***, ###: <i>P</i> < 0.001. Rd, radotinib; Das, dasatinib; ATRA, all-trans retinoic acid.</p

RhLIGHT enhances the viability and proliferation of human BM-MSCs.

<p>Cells were incubated with 0, 100, and 200 ng/mL rhLIGHT for 72 h. (A) Cell viability of BM-MSCs, as determined by MTS assay. (B) Expression of survival proteins and anti-apoptotic proteins, as determined by western blotting. (C) Cell proliferation of BM-MSCs, as determined by BrdU assay. (D) Cell cycle distribution of BM-MSCs, as determined by PI/RNase assay (E) Expression of cell cycle-related proteins, as determined by western blotting. The membrane was stripped and reprobed with anti-β-actin mAb to confirm equal loading. Data represent the mean ± SEM. Significantly different from the control cells (*); ***, <i>P</i> < 0.001.</p

Radotinib induces caspase-3 dependent apoptosis of CD11b⁺ cells differentiated from AML cells.

<p>Kasumi-1 cells were incubated with 0, 1, 5, and 10 μM of radotinib, and BMCs from a patient with AML were stimulated with 0, 1, 10 and 100 μM of radotinib for 72 h. Then the cells were harvested and used for measurements of the mitochondrial membrane potential using Dioc₆(3) dye, intracellular staining for cleaved caspase-3, or studies of the caspase-3 activity in CD11b⁺ cells, as described in Materials and Methods. (A) The percentage of Dioc₆(3)⁺ cells in the total population of CD11b⁺ cells. (B) The number of Dioc₆(3)⁺ cells among CD11b⁺ cells. (C) CD11b⁺cleaved caspase-3⁺ cells among Kasumi-1 cells. (D) CD11b⁺ cleaved caspase-3⁺ cells among BMCs isolated from an AML-3 patient. (E) The expression of Annexin V, CD11b, and cleaved caspase-3 induced by 5 μM radotinib in Kasumi-1 cells was monitored by FlowSight analysis. (F) Caspase-3 activity in CD11b⁺ Kasumi-1 cells. Data represent the mean ± SEM. Statistically significant differences from the DMSO-treated control (*) are denoted as follows. ***: <i>P</i> < 0.001. BF, bright field; cCas-3, cleaved caspase-3.</p

Characterization of bone marrow-derived MSCs (BM-MSCs).

<p>(A) Negative marker (CD34, CD45, and CD19) staining in BM-MSCs. (B) Positive marker (CD90, CD44, and CD105) staining in BM-MSCs. The level of expression of each marker was determined by FACS analysis. Filled histogram represents the isotype control (mouse IgG); open histogram represents each antigen. (C) Representative images of cells such as adipocytes, chondrocytes, and osteocytes undergoing differentiation via adipogenesis (Oil Red O staining), chondrogenesis (Alcian blue staining), and osteogenesis (Alizarin red staining), respectively. MSCs, mesenchymal stromal cells, MSCs; BM-MSCs, bone marrow-derived MSCs; P, passage number.</p

Information of patients.

<p>AML, acute myeloid leukemia; CML, chronic myeloid leukemia; BP, blast phase; CP, chronic phase.</p><p>Information of patients.</p