Up-Regulatory Effects of Curcumin on Large Conductance Ca2+-Activated K+ Channels.

Large conductance Ca2+-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+β1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms

Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts.

Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells

Hydrogen Sulfide Inhibits Transforming Growth Factor-β1-Induced EMT via Wnt/Catenin Pathway.

Hydrogen sulfide (H2S) has anti-fibrotic potential in lung, kidney and other organs. The exogenous H2S is released from sodium hydrosulfide (NaHS) and can influence the renal fibrosis by blocking the differentiation of quiescent renal fibroblasts to myofibroblasts. But whether H2S affects renal epithelial-to-mesenchymal transition (EMT) and the underlying mechanisms remain unknown. Our study is aimed at investigating the in vitro effects of H2S on transforming growth factor-β1 (TGF-β1)-induced EMT in renal tubular epithelial cells (HK-2 cells) and the associated mechanisms. The induced EMT is assessed by Western blotting analysis on the expressions of α-SMA, E-cadherin and fibronectin. HK-2 cells were treated with NaHS before incubating with TGF-β1 to investigate its effect on EMT and the related molecular mechanism. Results demonstrated that NaHS decreased the expression of α-SMA and fibronectin, and increased the expression of E-cadherin. NaHS reduced the expression of TGF-β receptor type I (TβR I) and TGF-β receptor type II (TβR II). In addition, NaHS attenuated TGF-β1-induced increase of β-catenin expression and ERK phosphorylation. Moreover, it inhibited the TGF-β1-induced nuclear translocation of ββ-catenin. These effects of NaHS on fibronectin, E-cadherin and TβR I were abolished by the ERK inhibitor U0126 or β-catenin inhibitor XAV939, or β-catenin siRNA interference. We get the conclusion that NaHS attenuated TGF-β1-induced EMT in HK-2 cells through both ERK-dependent and β-catenin-dependent pathways

Functional effect of actinin 4 on L-type calcium channel in osteoblast Ros 17/2.8 cells.

<p>(A) shRNA interference silenced the expression of actinin 4 in Ros 17/2.8 cells, and the effect was not exhibited on actin. (B) The representative traces of L-type calcium channel in control and actinin 4 shRNA group. (C) Current-voltage relationship of L-type calcium channel. The actinin 4 shRNA decreased L-type calcium channel currents in control (P<0.05, n = 6) and forskolin treated group (P<0.05, n = 6).</p

The interaction of actinin 4 and L-type calcium channel α_1c subunit with 20 μM forskolin for 24 h.

<p>Ros17/2.8 cells were transfected with either L-type calcium channel α_<b>1c</b> subunit alone or co-transfected with L-type calcium channel α_<b>1c</b> subunit and β_<b>3</b> subunit. The cell lysates were pulled down with GST-actinin 4 conjugated beads and then were subjected to western blot analysis, using anti-α_<b>1c</b> antibody. (A) The expression of actinin 4 in Ros17/2.8 cells. (B) The expression of β_<b>3</b> subunit in Ros17/2.8 cells. (C) The interaction of actinin 4 and L-type calcium channel α_<b>1c</b> subunit. The results showed that actinin 4 did not interact with L-type calcium channel α_<b>1c</b> subunit, and forskolin did not significantly affect the interaction of actinin 4 with α_<b>1c</b> subunit (n = 3).</p

Effect of forskolin on the surface expression of L-type calcium channel α_1c subunit inosteoblast Ros17/2.8 cells.

<p>(A) Cell surface expression of L-type calcium channel α_<b>1c</b> subunit of Ros17/2.8 cells is shown after treatment of 20 μM forskolin for 15 min. The quantitative data show the total (B) and surface (C) α_<b>1c</b> protein levels. There is no significant difference between these groups (n = 3, P>0.05). (D) Cell surface expression of L-type calcium channel α_<b>1c</b> subunit of Ros17 /2.8 cells is shown after treatment of 20 μM forskolin for 3 h and 24 h. The quantitative datas show the total (E) and surface (F) α_<b>1c</b> protein levels. There is also no difference between these groups (n = 4, P>0.05).</p

Curcumin increased BK protein stability via inhibition of degradation pathways.

<p>(A) Effect of curcumin on BK protein stability was examined with cycloheximide-chase assay. Western blot analysis showed that treatment with 10μM curcumin prolonged BK protein half-life. (B) Graphic representation of densitometric data showing the remaining BK protein level in Fig 7A (n = 3). (C) Effect of curcumin on BK protein in the presence and absence of proteasomal inhibitor, MG132. Western blot analysis showed that the increase of BK protein level was abolished by the proteasome inhibitor, MG132 (5 μM). Inhibitors were applied 1 h before the administration of curcumin. (D) Graphic representation of densitometric data of the BK protein level in Fig 7C (n = 3). * <i>p</i> < 0.05; n.s., not significant.</p

The interaction of actinin 4 and L-type calcium channel β₃ subunit with 20 μM forskolin treatment for 24 h in osteoblast Ros17/2.8 cells.

<p>Cells were transfected with L-type calcium channel β_<b>3</b> subunit. (A1) Sample was pulled down with GST-actinin 4 conjugated beads, and immunoblotted with anti-β_<b>3</b> subunit antibody. (A2) The quantitative ratio of pull-down β_<b>3</b> over the input β_<b>3</b> subunit (P<0.05, n = 6). (B1) Sample was immunoprecipitated with anti-β_<b>3</b> subunit antibody, and immunoblotted with anti-actinin 4 antibody. (B2) The quantitative ratio of IP actinin 4 over the input actinin 4 (P<0.05, n = 3). (C1) The cell lysates were treated with forskolin <i>in vitro</i> and then pulled down with GST-actinin 4 conjugated beads. Anti-β_<b>3</b> subunit antibody was used to detect the protein by western blot analysis. (C2) The quantitative ratio of pull-down β_<b>3</b> over the input β_<b>3</b> subunit <i>in vitro</i> (P<0.05, n = 3).</p

Effect of curcumin on BK protein abundance and BK mRNA in A7r5 cells.

<p>(A) Western blot analysis showed that treatment with curcumin at 0, 2.5, 5, 10, and 20 μM for 24h up-regulated the BK protein expression in A7r5 cells. * <i>p</i> <0.05 (n = 3). Graphic representative of densitometric data of the above total BK protein level are shown in (B). (C) Real-time RT-PCR for the measurement of BK mRNA levels with 10 μM curcumin treatment; the BK mRNA levels were normalized with GAPDH. The predicted amplicon size for BK and GAPDH is 103 bp and 271 bp, respectively. n.s., not significant (n = 3).</p

Effect of curcumin on exogenous BK (α) channel currents in HEK293 cells.

<p>(A) Representative whole cell current traces from HEK293 cells expressing BK channels before and after the perfusion of 5 μM curcumin (left) and the time course of the enhancement on BK (α) channels by of curcumin (right). With 3 μM free Ca²⁺ in the pipette solution, the holding voltage was controlled at -80 mV and BK currents were evoked by the pulse of +100 mV. (B) The dose-dependence curve of curcumin enhancing BK currents was fitted by the Hill equation (see “Data analysis”). The EC₅₀ value is 5.83±0.76 μM with a Hill coefficient of n = 1.71±0.30 (n = 8). (C) HEK293 cells were held at -80 mV, and the duration of 200 ms voltage steps were applied from -50 to +120 mV at 10 mV increments. Representative trace before treatment with curcumin was shown. (D) Representative trace manifests the effect of curcumin at 5 μM. (E) The plots of the normalized conductance were fitted well with Boltzmann function (“see Data analysis”). The voltage dependence of steady state activation curve was not shifted significantly in the presence of curcumin (n = 8). □ represents the curve of BK channels before exposure to curcumin. ○ represents the curve of BK channels after exposure to 5 μM curcumin. Δ represents the curve of BK channels after exposure to 20 μM curcumin. Representative whole cell current traces from HEK293 cells expressing BK channels before (F) and after (G) the perfusion of curcumin 5 μM for 24 hours. (H) The dose-dependence curve of enhanced BK current density by curcumin was fitted by the Hill equation (see “Data analysis”). The EC₅₀ value is 8.05±0.97 μM with a Hill co-efficient of n = 1.77±0.45 (n = 5). (I) Representative trace of BK currents and (J) the trace manifested the effect of curcumin at the concentration of 5 μM. (K) The plots of the normalized conductance were fitted well with Boltzmann function (“see Data analysis”). The voltage dependence of steady state activation curve was not shifted significantly in the presence of curcumin (n = 9). □ represents the curve of BK channels before exposure to curcumin. ○ represents the curve of BK channels after exposure to 5 μM curcumin. Δ represents the curve of BK channels after exposure to 20 μM curcumin.</p