DLK1 and DLK2, two non-canonical ligands of NOTCH receptors, differentially modulate the osteogenic differentiation of mesenchymal C3H10T1/2 cells

doi:10.1186/s40659-024-00561-7

. 2024 Oct 30;57(1):77.

doi: 10.1186/s40659-024-00561-7.

DLK1 and DLK2, two non-canonical ligands of NOTCH receptors, differentially modulate the osteogenic differentiation of mesenchymal C3H10T1/2 cells

María-Milagros Rodríguez-Cano^#¹, María-Julia González-Gómez^#², Eva-María Monsalve³, María-José M Díaz-Guerra³, Moustapha Kassem⁴, Jorge Laborda¹, María-Luisa Nueda⁵, Victoriano Baladrón⁶

Affiliations

¹ Biochemistry and Molecular Biology Branch, Department of Inorganic, Organic Chemistry and Biochemistry, Pharmacy School/IB-UCLM/Biomedicine Unit, University of Castilla-La Mancha/CSIC, Albacete, Spain.
² Biochemistry and Molecular Biology Branch, Department of Inorganic, Organic Chemistry and Biochemistry, ETSIAMB/IB-UCLM/Biomedicine Unit, University of Castilla-La Mancha/CSIC, Albacete, Spain.
³ Biochemistry and Molecular Biology Branch, Department of Inorganic, Organic Chemistry and Biochemistry, Medical School/IB-UCLM/Biomedicine Unit, University of Castilla-La Mancha/CSIC, Albacete, Spain.
⁴ Molecular Endocrinology Unit (KMEB), Department of Endocrinology and Metabolism, University Hospital of Odense and Danish Institute for Advanced Study, University of Southern Denmark, Odense, Denmark.
⁵ Biochemistry and Molecular Biology Branch, Department of Inorganic, Organic Chemistry and Biochemistry, Pharmacy School/IB-UCLM/Biomedicine Unit, University of Castilla-La Mancha/CSIC, Albacete, Spain. MariaLuisa.Nueda@uclm.es.
⁶ Biochemistry and Molecular Biology Branch, Department of Inorganic, Organic Chemistry and Biochemistry, Medical School/IB-UCLM/Biomedicine Unit, University of Castilla-La Mancha/CSIC, Albacete, Spain. Victoriano.Baladron@uclm.es.

^# Contributed equally.

PMID: 39473022
PMCID: PMC11523663
DOI: 10.1186/s40659-024-00561-7

DLK1 and DLK2, two non-canonical ligands of NOTCH receptors, differentially modulate the osteogenic differentiation of mesenchymal C3H10T1/2 cells

María-Milagros Rodríguez-Cano et al. Biol Res. 2024.

. 2024 Oct 30;57(1):77.

doi: 10.1186/s40659-024-00561-7.

Authors

Affiliations

¹ Biochemistry and Molecular Biology Branch, Department of Inorganic, Organic Chemistry and Biochemistry, Pharmacy School/IB-UCLM/Biomedicine Unit, University of Castilla-La Mancha/CSIC, Albacete, Spain.
² Biochemistry and Molecular Biology Branch, Department of Inorganic, Organic Chemistry and Biochemistry, ETSIAMB/IB-UCLM/Biomedicine Unit, University of Castilla-La Mancha/CSIC, Albacete, Spain.
³ Biochemistry and Molecular Biology Branch, Department of Inorganic, Organic Chemistry and Biochemistry, Medical School/IB-UCLM/Biomedicine Unit, University of Castilla-La Mancha/CSIC, Albacete, Spain.
⁴ Molecular Endocrinology Unit (KMEB), Department of Endocrinology and Metabolism, University Hospital of Odense and Danish Institute for Advanced Study, University of Southern Denmark, Odense, Denmark.
⁵ Biochemistry and Molecular Biology Branch, Department of Inorganic, Organic Chemistry and Biochemistry, Pharmacy School/IB-UCLM/Biomedicine Unit, University of Castilla-La Mancha/CSIC, Albacete, Spain. MariaLuisa.Nueda@uclm.es.
⁶ Biochemistry and Molecular Biology Branch, Department of Inorganic, Organic Chemistry and Biochemistry, Medical School/IB-UCLM/Biomedicine Unit, University of Castilla-La Mancha/CSIC, Albacete, Spain. Victoriano.Baladron@uclm.es.

^# Contributed equally.

PMID: 39473022
PMCID: PMC11523663
DOI: 10.1186/s40659-024-00561-7

Abstract

Background: C3H10T1/2 is a mesenchymal cell line capable of differentiating into osteoblasts, adipocytes and chondrocytes. The differentiation of these cells into osteoblasts is modulated by various transcription factors, such as RUNX2. Additionally, several interconnected signaling pathways, including the NOTCH pathway, play a crucial role in modulating their differentiation into mature bone cells. We have investigated the roles of DLK1 and DLK2, two non-canonical inhibitory ligands of NOTCH receptors, in the osteogenic differentiation of C3H10T1/2 cells.

Results: Our results corroborate existing evidence that DLK1 acts as an inhibitor of osteogenesis. In contrast, we demonstrate for the first time that DLK2 enhances this differentiation process. Additionally, our data suggest that NOTCH2, 3 and 4 receptors may promote osteogenesis, as indicated by their increased expression during this process, whereas NOTCH1 expression, which decreases during cell differentiation, might inhibit osteogenesis. Moreover, treatment with DAPT, a NOTCH signaling inhibitor, impeded osteogenic differentiation. We have confirmed the increase in ERK1/2 MAPK and p38 MAPK phosphorylation in C3H10T1/2 cells induced to differentiate to osteoblasts. Our new findings reveal increased ERK1/2 MAPK phosphorylation in differentiated C3H10T1/2 cells with a decrease in DLK1 expression or an overexpression of DLK2, which is coincident with the behavior of those transfectants where we have detected an increase in osteogenic differentiation. Additionally, p38 MAPK phosphorylation increases in differentiated C3H10T1/2 cells with reduced DLK1 levels.

Conclusions: Our results suggest that DLK1 may inhibit osteogenesis, while DLK2 may promote it, by modulating NOTCH signaling and the phosphorylation of ERK1/2 and p38 MAPK pathways. Given the established inhibitory effect of DLK proteins on NOTCH signaling, these new insights could pave the way for developing future therapeutic strategies aimed at treating bone diseases.

Keywords: DLK; ERK1/2 MAPK; Mesenchymal C3H10T1/2 cells; NOTCH; Osteogenesis; p38 MAPK.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

**Fig. 1**
Osteogenic differentiation in C3H10T1/2 cells induced by β-glycerophosphate, ascorbic acid, and all trans retinoic acid (ATRA). A Alkaline phosphatase staining illustrating the progression of osteoblastic differentiation in C3H10T1/2 cells over 1, 7, 14, and 21 days of induction. B ALP activity (U/L) in C3H10T1/2 cells over 1, 7, 14, and 21 days of osteogenesis induction. RT-qPCR analysis of the osteogenesis-specific marker in C3H10T1/2 cells. Relative mRNA expression levels of *Alpl* (C) *Col1a1* (D), *Runx2* (E), *Opn* (F), and *Ocn* (G) osteogenic markers were also measured in differentiated C3H10T1/2 cells over a period of 1, 7, 14, and 21 days of induction. Expression data were normalized against the constitutive ribosomal gene *Rplp0*. Expression levels are relative to day 1 in undifferentiated cells (set arbitrarily at 1) [horizontal line]. Absence of this line in some graphs is due to overlap with the horizontal axis due to scale adjustments. Data are presented as mean ± SD from a minimum of three independent assays, each performed in triplicate. Statistical significance was assessed using Student’s t-test (***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05)

**Fig. 2**
Influence of DAPT, a γ-secretase complex inhibitor, on the expression of osteogenic markers during the differentiation of C3H10T1/2 cells . A Representative images from alkaline phosphatase staining depict the contrast in osteogenic activity of undifferentiated and differentiated C3H10T1/2 cells over 1, 7, 14, and 21 days [D], in both the presence and absence of the DAPT inhibitor. B–F The figure further includes a RT-qPCR analysis of the relative mRNA expression levels of osteogenic markers *Alpl* (B), *Col1a1* (C), *Runx2* (D), *Opn* (E), and *Ocn* (F) in C3H10T1/2 cells undergoing osteoblastic differentiation with or without DAPT treatment at the same time points. The RT-qPCR data were normalized against the mRNA levels of the constitutive ribosomal gene *Rplp0*, and expression levels were calculated relative to day 1 in undifferentiated cells treated with DMSO (horizontal line). The absence of the horizontal line in some graphs is due to its overlap with the horizontal axis because of the vertical axis scale. Data are presented as mean ± SD from at least three independent experiments, each performed in triplicate. Statistical significance was determined using Student’s t-test (***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05), with non-significant results indicated as ns

**Fig. 3**
Characterization of *Dlk1* and *Dlk2* stable transfectant pools in C3H10T1/2 cells. RT-qPCR analysis of the relative mRNA expression levels of *Dlk1* in DLK1S and DLK1aS stable transfectant pools (A), and *Dlk2* in DLK2S and DLK2aS stable transfectant pools (B). C, D Representative Western blots and densitometric analyses illustrate DLK1 (50–60 kDa) (C) and DLK2 (40 kDa) (D) protein expression levels in these pools. E Global NOTCH signaling activity assessed by luciferase assay in DLK1 and DLK2 transfectants compared to control cells transfected with empty vector (V). The figure also presents RT-qPCR analysis of *Hes1* (F) and *Hey1* (G) mRNA expression levels in each stable transfectant pool. α-Tubulin was employed as a control for loading and sample quality in Western blot assays. Data from RT-qPCR assays were normalized against the constitutive ribosomal gene *Rplp0*, with expression levels calculated relative to cells stably transfected with the corresponding empty vector (set arbitrarily at 1) [horizontal line]. Data are presented as mean ± SD from a minimum of three independent assays performed in triplicate. Statistical significance was assessed using Student’s t-test (***p ≤ 0.001, and *p ≤ 0.05), and non-significant results are denoted as ns

**Fig. 4**
Alkaline phosphatase staining and analysis of osteogenic marker expression levels in *Dlk1* stable transfectant pools of C3H10T1/2 cells. A This part showcases representative images of alkaline phosphatase staining in cell culture wells. It includes both non-transfected and *Dlk1* stable transfectant pools of C3H10T1/2 cells that have undergone osteoblastic differentiation. The images capture the staining results at 1-, 7-, 14-, and 21-days [D] post-induction of osteogenic differentiation. The cultures include C3H10T1/2 non-transfected cells, cells transfected with empty vector control [V], and cells from the DLK1S and DLK1aS transfectant pools, providing a comparative view of alkaline phosphatase activity across different genetic modifications and stages of differentiation. In this figure, we also show a RT-qPCR analysis of the relative mRNA expression levels of key osteogenic markers in *Dlk1* sense (DLK1S) and antisense (DLK1aS) stable transfectant pools of C3H10T1/2 cells. The markers analyzed include *Alpl* (B), *Col1a1* (C), *Runx2* (D), *Opn* (E), and *Ocn* (F). The expression levels were measured in cells differentiated into osteoblasts over a period of 1-, 7-, 14-, and 21-days [D] post-induction of osteogenic differentiation. The RT-qPCR data were normalized against the mRNA levels of the constitutive ribosomal gene *Rplp0*, with expression levels calculated relative to day 1 in cells stably transfected with the empty vector (set arbitrarily at 1) [horizontal line]. The absence of the horizontal line in some graphs is due to its overlap with the horizontal axis resulting from the vertical axis scale adjustments. Results are shown as mean ± SD from a minimum of three independent assays, each performed in triplicate. Statistical significance was assessed using Student’s t-test (***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05), and non-significant results are denoted as ns

**Fig. 5**
Alkaline phosphatase staining and analysis of osteogenic marker expression levels in *Dlk2* stable transfectant pools of C3H10T1/2 cells. A This part showcases representative images of alkaline phosphatase staining in cell culture wells. It includes both transfected and *Dlk2* stable transfectant pools of C3H10T1/2 cells that have undergone osteoblastic differentiation. The images capture the staining results at 1-, 7-, 14-, and 21-days [D] post-induction of osteogenic differentiation. The cultures include C3H10T1/2 non-transfected cells, cells transfected with empty vector control [V], and cells from the DLK2S and DLK2aS transfectant pools, providing a comparative view of alkaline phosphatase activity across different genetic modifications and stages of differentiation. In this figure, we also show a RT-qPCR analysis of the relative mRNA expression levels of key osteogenic markers in *Dlk2* sense (DLK2S) and antisense (DLK2aS) stable transfectant pools of C3H10T1/2 cells. The markers analyzed include *Alpl* (B), *Col1a1* (C), *Runx2* (D), *Opn* (E), and *Ocn* (F). The expression levels were measured in cells differentiated into osteoblasts over a period of 1-, 7-, 14-, and 21-days [D] post-induction of osteogenic differentiation. The RT-qPCR data were normalized against the mRNA expression levels of the ribosomal gene *Rplp0*. Expression levels for each marker were compared with values from cells stably transfected with the empty vector on day 1, set as a baseline (horizontal line). The absence of the horizontal line in some graphs is due to its overlap with the horizontal axis, resulting from the scaling of the vertical axis. Data are presented as mean ± SD from at least three independent assays, each performed in triplicate. Statistical significance was evaluated using Student’s t-test (***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05), and non-significant results are indicated as ns

**Fig. 6**
ERK1/2 MAPK phosphorylation dynamics in *Dlk1* and *Dlk2* stable transfectant pools during osteogenic differentiation. A Representative Western blot images displaying the phosphorylation levels of ERK1/2 MAPK (42–44 kDa) in *Dlk1* and *Dlk2* stable transfectant pools of C3H10T1/2 cells at various stages of osteogenic differentiation (1, 7, 14, and 21 days [D]). B Densitometric analysis quantifying ERK1/2 MAPK phosphorylation levels in *Dlk1* and *Dlk2* transfectants during osteogenic differentiation. The data are normalized to total ERK1/2 MAPK expression, serving as control for loading and sample integrity. The baseline phosphorylation level was set using data from non-differentiated cells stably transfected with the empty vector on day 1 (indicated by the horizontal line). The densitometric results are presented as the mean ± SD from at least three independent experiments, each performed in triplicate. Statistical analysis was conducted using Student’s t-test to compare each transfectant pool at different time points against the day 1 baseline, with significance levels marked as ***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05. Results not reaching statistical significance are denoted as ns

**Fig. 7**
p38 MAPK phosphorylation dynamics in *Dlk1* and *Dlk2* stable transfectant pools during osteogenic differentiation. A Representative Western blot images showcase the phosphorylation levels of p38 MAPK phosphorylation in *Dlk1* and *Dlk2* stable transfectant pools of C3H10T1/2 cells at various stages of osteogenic differentiation (1, 7, 14, and 21 days [D]). B Densitometric analysis provides a quantitative assessment of p38 MAPK phosphorylation in DLK1 and DLK2 transfectant pools. The data are normalized to total p38 MAPK expression, serving as controls for loading and sample integrity. The baseline phosphorylation level was set using data from non-differentiated cells stably transfected with the empty vector on day 1 (indicated by the horizontal line). The densitometric results are presented as the mean ± SD from at least three independent experiments, each performed in triplicate. Statistical analysis was conducted using Student’s t-test to compare each transfectant pool at different time points against the day 1 baseline, with significance levels marked as ***p ≤ 0.001, and **p ≤ 0.01. Results not reaching statistical significance are denoted as ns

**Fig. 8**
A Effect of osteogenic treatment (10 mM β-glycerol-phosphate (β-GP), 50 μg/ml ascorbic acid (AA), and 1 μM all-trans retinoic acid (ATRA)) on the expression levels of osteogenic markers and *Notch* family genes, as well as the phosphorylation levels of ERK1/2 and p38 MAPK, in non-transfected mesenchymal C3H10T1/2 cells. B Impact of treatment with DAPT, a NOTCH signaling inhibitor (GSI), on the osteogenesis of non-transfected mesenchymal C3H10T1/2 cells, and the effect of DLK1 and DLK2, two inhibitory ligands of NOTCH receptors, on the osteogenesis of mesenchymal C3H10T1/2 cells transfected with plasmids that overexpress DLK1 or DLK2 proteins (DLK1S or DLK2S: DLK1 or DLK2 in sense orientation) or plasmids that downregulate DLK1 or DLK2 proteins (DLK1aS or DLK2aS: DLK1 or DLK2 in antisense orientation). Red (-) symbol indicates inhibition of osteogenesis. Green (+) symbol indicates activation of osteogenesis. Green up arrow means increased gene expression. Red down arrow means decreased gene expression

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References

1. Lin G, Hankenson K. Integration of BMP, wnt, and notch signaling pathways in osteoblast differentiation. J Cell Biochem. 2011;112:3491–501. - PMC - PubMed
1. Brou C, et al. A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell. 2000;5(2):207–16. - PubMed
1. Lai EC. Notch cleavage: Nicastrin helps Presenilin make the final cut. Curr Biol. 2002;12(6):R200–2. - PubMed
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1. Laborda J. 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein PO. Nucleic Acids Res. 1991;19(14):3998. - PMC - PubMed

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ISBPLY/17/180501/000316/The Health Council of the Regional Government of Castilla-La Mancha (Spain), supported by the Fondo Europeo de Desarrollo Regional (FEDER).

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[1] Lin G, Hankenson K. Integration of BMP, wnt, and notch signaling pathways in osteoblast differentiation. J Cell Biochem. 2011;112:3491–501. - PMC - PubMed

[2] Lin G, Hankenson K. Integration of BMP, wnt, and notch signaling pathways in osteoblast differentiation. J Cell Biochem. 2011;112:3491–501. - PMC - PubMed

[3] Brou C, et al. A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell. 2000;5(2):207–16. - PubMed

[4] Brou C, et al. A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell. 2000;5(2):207–16. - PubMed

[5] Lai EC. Notch cleavage: Nicastrin helps Presenilin make the final cut. Curr Biol. 2002;12(6):R200–2. - PubMed

[6] Lai EC. Notch cleavage: Nicastrin helps Presenilin make the final cut. Curr Biol. 2002;12(6):R200–2. - PubMed

[7] Barrick D, Kopan R. The Notch transcription activation complex makes its move. Cell. 2006;124(5):883–5. - PubMed

[8] Barrick D, Kopan R. The Notch transcription activation complex makes its move. Cell. 2006;124(5):883–5. - PubMed

[9] Laborda J. 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein PO. Nucleic Acids Res. 1991;19(14):3998. - PMC - PubMed

[10] Laborda J. 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein PO. Nucleic Acids Res. 1991;19(14):3998. - PMC - PubMed

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DLK1 and DLK2, two non-canonical ligands of NOTCH receptors, differentially modulate the osteogenic differentiation of mesenchymal C3H10T1/2 cells

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DLK1 and DLK2, two non-canonical ligands of NOTCH receptors, differentially modulate the osteogenic differentiation of mesenchymal C3H10T1/2 cells

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Conflict of interest statement

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