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. 2009 Oct 15;108(3):577-88.
doi: 10.1002/jcb.22289.

Interleukin-6 maintains bone marrow-derived mesenchymal stem cell stemness by an ERK1/2-dependent mechanism

Katie L Pricola  1 , Nastaran Z Kuhn, Hana Haleem-Smith, Yingjie Song, Rocky S Tuan
Affiliations

Interleukin-6 maintains bone marrow-derived mesenchymal stem cell stemness by an ERK1/2-dependent mechanism

Katie L Pricola et al. J Cell Biochem. .

Abstract

Adult human mesenchymal stem cells (MSCs) hold promise for an increasing list of therapeutic uses due to their ease of isolation, expansion, and multi-lineage differentiation potential. To maximize the clinical potential of MSCs, the underlying mechanisms by which MSC functionality is controlled must be understood. We have taken a deconstructive approach to understand the individual components in vitro, namely the role of candidate "stemness" genes. Our recent microarray gene expression profiling data suggest that interleukin-6 (IL-6) may contribute to the maintenance of MSCs in their undifferentiated state. In this study, we showed that IL-6 gene expression is significantly higher in undifferentiated MSCs as compared to their chondrogenic, osteogenic, and adipogenic derivatives. Moreover, we found that MSCs secrete copious amounts of IL-6 protein, which decreases dramatically during osteogenic differentiation. We further evaluated the role of IL-6 for maintenance of MSC "stemness," using a series of functional assays. The data showed that IL-6 is both necessary and sufficient for enhanced MSC proliferation, protects MSCs from apoptosis, inhibits adipogenic and chondrogenic differentiation of MSCs, and increases the rate of in vitro wound healing of MSCs. We further identified ERK1/2 activation as the key pathway through which IL-6 regulates both MSC proliferation and inhibition of differentiation. Taken together, these findings show for the first time that IL-6 maintains the proliferative and undifferentiated state of bone marrow-derived MSCs, an important parameter for the optimization of both in vitro and in vivo manipulation of MSCs.

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Figures

Figure 1
Figure 1. IL-6 gene expression is significantly lower in differentiated vs. undifferentiated MSCs
(A) Graph showing IL-6 gene expression of undifferentiated MSCs (MSC) and MSCs that have undergone osteogenic (Osteo), adipogenic (Adipo), and chondrogenic (Chondro) differentiation. Data are normalized against GAPDH. Compared to undifferentiated MSCs, IL-6 mRNA levels were 5-fold and 2-fold lower in osteogenic cells at days 7 and 14 of differentiation; 9-fold and 3-fold lower in adipogenic cells at day 7 and day 14 of differentiation; and 18-fold and 20-fold lower in chondrogenic cells at day 7 and day 14 of differentiation (ANOVA, all compared to MSC: * p < 0.05, ** p < 0.01, *** p < 0.001). (B) Graph showing IL-6 protein secretion levels as measured by ELISA for undifferentiated MSCs and osteogenic differentiated MSCs. After 7 days of osteogenic differentiation, there was a significant 2-fold drop in IL-6 secretion levels compared to undifferentiated MSC controls (t-test: *** p < 0.001).
Figure 2
Figure 2. IL-6 enhances proliferation of undifferentiated MSCs
(A) Compared to non-stimulated controls, undifferentiated MSCs grown in the presence of IL-6 (10 ng/mL) had a 32% ± 3, 44% ± 8, and 62% ± 14 increase in cell number after 1, 2, and 3 days of growth, respectively. (B) Graph showing results from the MTT assay. Compared to non-stimulated controls, undifferentiated MSCs previously maintained in an IL-6 rich environment proliferated at a higher rate up to 2 days following removal of IL-6 stimulation. (C) Following siRNA knockdown, real-time RT-PCR analysis of IL-6 transcript levels revealed an average 60% 90% decrease in IL-6 expression compared to transfected controls up to 5 days post transfection. (D) Loss of IL-6 expression decreased MSC proliferation by almost 50% at both 2 and 3 days post-transfection. A-D: Student’s t-test analysis, * p < 0.05, ** p < 0.01.
Figure 3
Figure 3. IL-6 protects MSCs from serum starvation-induced apoptosis
(A) TUNEL: Apoptosis was evaluated using TUNEL staining in undifferentiated MSCs grown in serum-free media for 72 h with or without IL-6. IL-6 treated populations exhibited fewer apoptotic cells compared to untreated controls. DAPI staining revealed IL-6 treated cells had smaller, more pyknotic nuclei and TUNEL staining was more intense in MSCs serum-starved and treated with IL-6. Magnification: 20X. (B) Cell counts: For each condition, total numbers of TUNEL-positive cells were counted and a ratio of the means was calculated. The mean ratio of apoptotic cells in serum-free conditions to apoptotic cells in IL-6 supplemented conditions was 3.5: 1 (t-test: * p < 0.05). (C) Microarray gene expression analysis: Expression levels of pro-apoptotic genes decreased upon treatment with IL-6 for 72 h, only significantly for CD27 and TRAF2.
Figure 4
Figure 4. IL-6 inhibits adipogenic differentiation of MSCs
(A) Graph showing real-time RT-PCR results of LPL and (B) FABP4 gene expression normalized to GAPDH in MSCs induced to undergo adipogenesis without (Control) or with IL-6 treatment for 14 or 21 days. In adipocytes differentiated in the presence of IL-6, LPL expression decreased by 20% at day 14 and 50% by day 21, and FABP4 gene expression decreased by 20% at day 21 (N=3). (C) Oil Red O staining is shown to reveal the presence of lipid droplets, indicating adipogenic differentiation. Images were taken at 20x magnification. (D) Graph showing quantification of Oil Red O staining, which reveals that IL-6 treatment significantly decreased lipidization. (E) Graph showing real-time RT-PCR results of LPL and (F) FABP4 gene expression normalized to GAPDH in MSCs pre-treated with IL-6 for 48 h prior to adipogenic differentiation for 18 d. A, D-F: Student’s t-test analysis, * p < 0.05, ** p < 0.01.
Figure 5
Figure 5. IL-6 does not affect osteogenic differentiation of MSCs
(A) Graph showing real-time RT-PCR results of Alkaline Phosphatase (ALP) gene expression at day 7 and (B) Osteocalcin (OC) gene expression at day 21, both normalized to GAPDH in MSCs induced to undergo osteogenesis without (Control) or with IL-6 treatment. A, B: Student’s t-test analysis, * p < 0.05, ** p < 0.01. (C) Immunohistochemical staining of ALP and Alizarin Red, both showing no difference between Control and +IL-6 treated cells. Images were taken at 4x magnification. (D) ALP activity assay results of osteogenic differentiated MSCs in the absence (Control) or presence of IL-6. Data are normalized to total DNA content. (E) Graph showing quantification of Alizarin Red staining.
Figure 6
Figure 6. IL-6 treatment suppresses chondrogenic differentiation of MSCs
(A) Graph showing real-time RT-PCR results of Collagen II (COL2A1) gene expression normalized to GAPDH at day 21 of differentiation in MSCs induced to undergo chondrogenesis without (Control) or with IL-6 treatment (N=4). (B) Alcian Blue immunohistochemical staining, showing decreased glycosaminoglycan expression in chondrogenic-differentiated pellets treated with IL-6. Images were taken at 4x magnification. (C) Consistent with the qualitative observations above, the sGAG assay results show a significant decrease in sulfated glycosaminoglycan formation in chondrogenic-differentiated MSCs treated with IL-6 for 21 days. Data are normalized to total DNA content. A, C: Student’s t-test analysis, * p < 0.05.
Figure 7
Figure 7. IL-6 accelerates in vitro wound healing in MSCs
(A) Wound healing: Confluent, undifferentiated MSCs were wounded and defect closure was observed over 18 h either in the absence (Control) or presence of IL-6. Defects were imaged at the time of wounding (0 h) and then at 6 h intervals for 18 h. MSCs exposed to IL-6 after initial wounding showed increased capacity for in vitro healing. Images were taken at 5x magnification. (B) Quantitative analysis: The remaining wound area in the presence of IL-6 at 6 h post-injury was approximately 15% less than that of untreated MSCs, at 12 h post-injury was approximately 33% less than untreated MSCs, and at 18 h was approximately 24% less than untreated MSCs (t-test: * p < 0.05).
Figure 8
Figure 8. IL-6 mediated effects in MSCs are dependent upon ERK1/2 activation
Undifferentiated MSCs were serum-starved for 12 h and then stimulated with IL-6 (10 ng/mL) for 0–60 min. (A) STAT3: IL-6 stimulation did not induce activation of STAT3 in undifferentiated MSCs. Activated STAT3 (STAT3 (P)) was only observed in the positive control lysates, while total STAT3 levels remained constant with IL-6 treatment. Actin is shown as a loading control. (B) ERK1/2: Serum-starved MSCs have baseline activation of ERK1/2 (ERK1/2 (P)), which increased after 5 min of IL-6 stimulation, before returning to baseline levels at 60 min. Actin is shown as a loading control. (C) U0126 inhibition: IL-6-induced ERK1/2 activation (ERK1/2 (P) in undifferentiated MSCs was inhibited by U0126. Actin is shown as a loading control. (D-G) Effect of ERK1/2 inhibition: (D) IL-6 alone significantly increased proliferation over controls, while IL-6 stimulation in the presence of U0126 failed to induce an increase in proliferation (N=3). (E, F) LPL and FABP4 mRNA levels were decreased by day 14 in cells differentiated after exposure to IL-6; LPL and FABP4 expression was increased in cells differentiated after exposure to IL-6+U0126 (N=3). (E) COL2A1 gene expression in MSCs pre-treated with vehicle (Control), IL-6, or IL-6+U0126. All real-time RT-PCR data shown are normalized against GAPDH (ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001).
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