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. 2016 Dec 20;7(51):84118-84127.
doi: 10.18632/oncotarget.11527.

IPMK and β-catenin mediate PLC-β1-dependent signaling in myogenic differentiation

Giulia Ramazzotti  1 , Anna Maria Billi  1 , Lucia Manzoli  1 , Cristina Mazzetti  1 , Alessandra Ruggeri  1 , Christophe Erneux  2 , Seyun Kim  3 , Pann-Ghill Suh  4 , Lucio Cocco  1 , Irene Faenza  1
Affiliations

IPMK and β-catenin mediate PLC-β1-dependent signaling in myogenic differentiation

Giulia Ramazzotti et al. Oncotarget. .

Abstract

In previous studies, we have reported that phospholipase C (PLC)-β1 plays a crucial role in myogenic differentiation and we determined the importance of its catalytic activity for the initiation of this process. Here we define the effectors that take part to its signaling pathway. We show that the Inositol Polyphosphate Multikinase (IPMK) is able to promote myogenic differentiation since its overexpression determines the up-regulation of several myogenic markers. Moreover, we demonstrate that IPMK activates the same cyclin D3 promoter region targeted by PLC-β1 and that IPMK-induced promoter activation relies upon c-jun binding to the promoter, as we have shown previously for PLC-β1. Furthermore, our data shows that IPMK overexpression causes an increase in β-catenin translocation and accumulation to the nuclei of differentiating myoblasts resulting in higher MyoD activation. Finally, we describe that PLC-β1 overexpression determines too an increase in β-catenin translocation and that PLC-β1, IPMK and β-catenin are mediators of the same signaling pathway since their overexpression results in cyclin D3 and myosin heavy chain (MYH) induction.

Keywords: IPMK; inositol phosphates; myogenic differentiation; phospholipase C-β1; β-catenin.

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Conflict of interest statement

CONFLICTS OF INTEREST

None.

Figures

Figure 1
Figure 1. IPMK overexpression promotes myogenic differentiation
(A): IPMK expression was evaluated by real-time PCR in C2C12 cells in growing medium (GM) and after 24, 48 and 72 h culture in differentiation medium (respectively 24 DM, 48 DM, 72 DM). (B), (C) and (D): IPMK was overexpressed in C2C12 cells and the expression of myogenic markers was evaluated in growing cells (GM), 24 h, and 48 h after switching to differentiation medium (24 DM and 48 DM). The expression of IPMK, myogenin, cyclin D3 (panel B) and cyclin D1 (panel C) was evaluated by real –time PCR. Data are from three independent experiments, *p < 0.05 vs corresponding ctrl sample. The expression of myosin heavy chain (MYH), cyclin D3, myogenin, and DDK-tagged IPMK was evaluated by Western blot using tubulin as loading control (panel D). Data are from three independent sets of experiments.
Figure 2
Figure 2. Effects of IPMK on cyclin D3 promoter activation
C2C12 cells were co-transfected with an empty vector (mock) or a vector coding for IPMK (IPMK) and either with a reporter vector coding for human growth hormone (hGH) under control of a fragment of cyclin D3 promoter (pD3-957) or with the same vector bearing a mutation in c-jun consensus sequence in the promoter region (pD3-957 mut). hGH production was evaluated in growing cells (GM) and 24 h after the induction of differentiation (DM). Data are from three independent experiments, *p < 0.05 vs corresponding mock sample and °p < 0.05 vs corresponding pD3-957 sample.
Figure 3
Figure 3. IPMK promotes β-catenin nuclear accumulation
C2C12 cell were transfected with an empty vector (Ctrl) or DDK-tagged IPMK vector (ovIpmk). Cells were grown in growing medium (GM) or in differentiation medium (48 DM) for 48 h and (A) nuclear extracts were tested for β-catenin and Myo-D expression. Histone was used as loading control. (B) IPMK expression was tested on whole cell lysate from the same samples. Data are from three independent experiments.
Figure 4
Figure 4. Effects of PLC-β1, β-catenin and IPMK overexpression on myogenic differentiation
C2C12 cells were transfected with and empty vector (Mock) or a vector coding for PLC-β1 (ov-β1, panel A and C) or for β-catenin (ovβcat, panel B and C) or DDK-tagged IPMK (ovIMPK, panel C). After 48 h of differentiation, growing cells (GM) and differentiating cells (48 DM) were harvested. Nuclear extracts were tested for β-catenin (panel A) and cyclin D3 (panel B) expression and histone was used as loading control. Whole cell lysate (panel C) were tested for myosin heavy chain expression (MYH) and tubulin was used as loading control. Data are from three independent experiments.
Figure 5
Figure 5. Effects of IPMK mutants' overexpression on myogenic differentiation
C2C12 cell were transfected with an empty vector (Mock) or vectors coding for wild type IPMK (ovIpmk), IPMK kinase-dead mutant (ovSA), IPMK mutated in the inositol-binding site (ovKA) or IPMK double mutant (ovKA/SA). 24 h after transfection growth medium was switched to differentiation medium (DM). (A) 24 h after changing the medium to DM, IPMK, myogenin and cyclin D3 expression was evaluated by real –time PCR. Data are from three independent experiments, *p < 0.05 vs mock sample. (B) 72 h after changing the medium to DM, whole cell lysates were tested for myosin heavy chain, β-catenin and cyclin D3 expression. Tubulin was used as loading control.
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