GDF-15 enhances intracellular Ca2+ by increasing Cav1.3 expression in rat cerebellar granule neurons

Abstract

GDF-15 (growth/differentiation factor 15) is a novel member of the TGF (transforming growth factor)-β superfamily that has critical roles in the central and peripheral nervous systems. We reported previously that GDF-15 increased delayed rectifier outward K(+) currents and Kv2.1 α subunit expression through TβRII (TGF-β receptor II) to activate Src kinase and Akt/mTOR (mammalian target of rapamycin) signalling in rat CGNs (cerebellar granule neurons). In the present study, we found that treatment of CGNs with GDF-15 for 24 h increased the intracellular Ca(2+) concentration ([Ca(2+)]i) in response to membrane depolarization, as determined by Ca(2+) imaging. Whole-cell current recordings indicated that GDF-15 increased the inward Ca(2+) current (ICa) without altering steady-state activation of Ca(2+) channels. Treatment with nifedipine, an inhibitor of L-type Ca(2+) channels, abrogated GDF-15-induced increases in [Ca(2+)]i and ICa The GDF-15-induced increase in ICa was mediated via up-regulation of the Cav1.3 α subunit, which was attenuated by inhibiting Akt/mTOR and ERK (extracellular-signal-regulated kinase) pathways and by pharmacological inhibition of Src-mediated TβRII phosphorylation. Given that Cav1.3 is not only a channel for Ca(2+) influx, but also a transcriptional regulator, our data confirm that GDF-15 induces protein expression via TβRII and activation of a non-Smad pathway, and provide novel insight into the mechanism of GDF-15 function in neurons.

Keywords: Akt/mTOR; Cav1.3; ERK; GDF-15; TβRII; [Ca2+]i.

Figure 1. Effect of GDF-15 on [Ca²⁺]_i induced by high K⁺ in rat CGNs

(A) Intracellular Ca²⁺ imaging of control and GDF-15-treated CGNs before (Base) and after (HK) depolarization by acute perfusion with 27 mM K⁺. Changes in fura 2 fluorescence excitation ratios with increasing [Ca²⁺]_i are depicted as a colour gradient from purple to red. Scale bar, 50 μm. (B) Changes in [Ca²⁺]_i upon application of a depolarizing stimulus, as measured by quantification of fluorescence excitation ratios. The arrow represents a 30-s perfusion with a depolarizing solution of 27 mM K⁺. (C) Statistical analysis of [Ca²⁺]_i induced by high K⁺ in the presence or absence of GDF-15. Results are means±S.E.M. *P<0.05 for the two groups connected with a straight line. Ctrl, control.

Figure 2. Effect of GDF-15 on I_Ca amplitude and steady-state Ca²⁺ channel activation

(A) Representative traces of control and GDF-15-treated CGNs. I_Ca was elicited by depolarization to 10 mV from a holding potential of −80 mV. (B) Statistical analysis of the effect of GDF-15 on I_Ca amplitude. Results are means±S.E.M. *P<0.05 for two groups connected with a straight line. (C) Representative traces obtained with a steady-state voltage protocol of control and GDF-15-treated CGNs. I_Ca was elicited by 200-ms depolarizing pulses from a holding potential of −80 mV to between −60 and +40 mV in 10-mV steps at 10-s intervals. (D) Voltage-dependent activation curves of I_Ca. *P<0.05 compared with corresponding control. (E) Steady-state activation curves of I_Ca obtained by plotting normalized conductance as a function of command potential. Data points were fitted using the Boltzmann function. Results are means±S.E.M.

Figure 3. Effect of nifedipine on the increase in [Ca²⁺]_i elicited by high K⁺ in CGNs with or without GDF-15 treatment

(A) Ca²⁺ imaging before and after depolarization by application of a 27-mM K⁺ solution in GDF-15-treated CGNs in the presence or absence of nifedipine. Scale bar, 50 μm. (B) Changes in [Ca²⁺]_i upon application of a depolarizing stimulus, as measured by quantification of fluorescence excitation ratios. Each arrow represents a 30-s perfusion with a depolarizing 27-mM K⁺ solution. (C) Statistical analysis of [Ca²⁺]_i in control and GDF-15-treated CGNs in the presence or absence of nifedipine. Results are means±S.E.M. *P<0.05 for the two groups connected with a straight line. Ctrl, control.

Figure 4. Effect of nifedipine on I_Ca in control and GDF-15-treated CGNs

(A) Representative traces obtained with a steady-state voltage protocol of control and GDF-15-treated CGNs in the presence or absence of nifedipine. (B) I–V curve of I_Ca. Results were obtained from six independent experiments and are means±S.E.M. *P<0.05. (C) Representative traces of control and GDF-15-treated CGNs in the presence or absence of nifedipine. I_Ca was elicited with depolarizing pulses to 10 mV from a holding potential of −80 mV. (D) Statistical analysis of the effect of nifedipine on I_Ca. Results are means±S.E.M. *P<0.05 for the two groups connected with a straight line. Ctrl, control.

Figure 5. Effect of GDF-15 on Ca_v1.2 and Ca_v1.3 expression in CGNs

(A) Statistical analyses of Ca_v1.2 and Ca_v1.3 mRNA levels detected using qPCR. CGNs were incubated with GDF-15 from 15 min to 36 h. (B) Western blot and statistical analyses of the effect of GDF-15 on Ca_v1.2 and Ca_v1.3 expression in CGNs. (C) Western blot and statistical analyses of Ca_v1.3 levels in CGNs after incubation with GDF-15 for 15 mins to 36 hrs. (D) Statistical analyses of the effect of GDF-15 on Ca_v1.3 promoter expression in CGNs determined by luciferase reporter assays. Promoter information is illustrated. Results are means±S.E.M. *P<0.05 for the two groups connected with a straight line. Ctrl, control.

Figure 6. Effect of Akt/mTOR and ERK pathway inhibition on the GDF-15-induced increase in Ca_v1.3 α subunit expression

(A and B) Western blot and statistical analyses of the effects of the Akt inhibitor LY294002 and mTOR inhibitor rapamycin (A) and the MEK inhibitor U0126 (B) on GDF-15-induced up-regulation of Ca_v1.3 protein levels. Results are means±S.E.M. *P<0.05 for the two groups connected with a straight line.

Figure 7. Effects of TβRI and TβRI/TβRII inhibitors on the GDF-15-induced increase in Ca_v1.3 protein level and gene promoter expression

(A–C) Western blot and statistical analyses of the effects of TβRI inhibitors (PP1 and SB431542) (A and B) and TβRI/TβRII dual inhibitor (LY2109761) (C) on GDF-15-induced up-regulation of Ca_v1.3 protein levels. (D) Statistical analyses of the effect of the effects of SB431542, LY2109761 and U0126 on GDF-15-induced up-regulation of Ca_v1.3 promoter expression in CGNs determined by luciferase reporter assays. Results are means±S.E.M. *P<0.05 for the two groups connected with a straight line.