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Uncoupling of ER-mitochondrial calcium communication by transforming growth factor-beta - PubMed

Uncoupling of ER-mitochondrial calcium communication by transforming growth factor-beta

Pál Pacher et al. Am J Physiol Renal Physiol. 2008 Nov.

Abstract

Transforming growth factor-beta (TGF-beta) has been implicated as a key factor in mediating many cellular processes germane to disease pathogenesis, including diabetic vascular complications. TGF-beta alters cytosolic [Ca2+] ([Ca2+]c) signals, which in some cases may result from the downregulation of the IP3 receptor Ca2+ channels (IP3R). Ca2+ released by IP3Rs is effectively transferred from endoplasmic reticulum (ER) to the mitochondria to stimulate ATP production and to allow feedback control of the Ca2+ mobilization. To assess the effect of TGF-beta on the ER-mitochondrial Ca2+ transfer, we first studied the [Ca2+]c and mitochondrial matrix Ca2+ ([Ca2+]m) signals in single preglomerular afferent arteriolar smooth muscle cells (PGASMC). TGF-beta pretreatment (24 h) decreased both the [Ca2+]c and [Ca2+]m responses evoked by angiotensin II or endothelin. Strikingly, the [Ca2+]m signal was more depressed than the [Ca2+]c signal and was delayed. In permeabilized cells, TGF-beta pretreatment attenuated the rate but not the magnitude of the IP(3)-induced [Ca2+]c rise, yet caused massive depression of the [Ca2+]m responses. ER Ca2+ storage and mitochondrial uptake of added Ca2+ were not affected by TGF-beta. Also, TGF-beta had no effect on mitochondrial distribution and on the ER-mitochondrial contacts assessed by two-photon NAD(P)H imaging and electron microscopy. Downregulation of both IP3R1 and IP3R3 was found in TGF-beta-treated PGASMC. Thus, TGF-beta causes uncoupling of mitochondria from the ER Ca2+ release. The sole source of this would be suppression of the IP3R-mediated Ca2+ efflux, indicating that the ER-mitochondrial Ca2+ transfer depends on the maximal rate of Ca2+ release. The impaired ER-mitochondrial coupling may contribute to the vascular pathophysiology associated with TGF-beta production.

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Figures

Fig. 1.
Fig. 1.

Effect of transforming growth factor (TGF)-β pretreatment (50 nM for 24 h) on angiotensin II (AII)- and thapsigargin (Tg)-induced cytosolic Ca2+ signals in intact rat preglomerular afferent arteriolar smooth muscle cells (PGASMCs). A and B: [Ca2+]c increase is shown by a green to red shift in the overlay of the green (excited at 380 nm) and red (excited at 340 nm) fura2 fluorescence images. Cells were sequentially stimulated with 2 and 100 nM AII. Time courses of the fluorescence ratio of 340- and 380-nm excitations (R340/380) calculated for the single cells marked by the numbers are shown in the graphs. C: mean time courses of [Ca2+]c signal evoked by Tg (2 μM) in naive (red) and TGF-β-pretreated (green) PGASMC. The data are representative of experiments repeated at least 5 times.

Fig. 2.
Fig. 2.

Propagation of AII-induced [Ca2+]c signals to the mitochondria in intact PGASMCs. Cells were loaded with both Mito Tracker Green (i images, shown in green) and rhod2 (ii images, shown in red). Following stimulation with 100 nM AII, there is a large increase in rhod2 fluorescence (bottom ii image, shown in red), which is colocalized with MitoTracker Green (overlaid image iii, shown in yellow). Notably, the images shown at the bottom were taken 1–2 min after the stimulation. By then, the [Ca2+]c signal had already decayed, thus above the nuclear region no visible increase of rhod2 fluorescence appears.

Fig. 3.
Fig. 3.

Effect of TGF-β pretreatment (50 nM for 24 h) on 2 and 100 nM AII-induced cytosolic and mitochondrial calcium signals. The cytosolic and mitochondrial calcium signals were measured in rhod2-loaded cells. Difference images (increase visualized in purple) and corresponding graphs show that in naive cells (A) stimulation evokes rapid cytosolic (green traces) and closely coupled mitochondrial (red traces) [Ca2+] signals, while in TGF-β-pretreated cells (B) there is a considerable delay and decrease both of cytosolic and mitochondrial [Ca2+] signals. [Ca2+]c traces were taken from the nuclear regions, and [Ca2+]m from regions showing mitochondrial structure after the stimulation. Note the difference in the time scale of the images of control and TGF-β-pretreated cells.

Fig. 4.
Fig. 4.

Propagation of the IP3-induced [Ca2+]c signals to the mitochondria in permeabilized PGASMC. Bottom: difference images and corresponding graphs (green traces) show IP3-induced rapid [Ca2+]c rise (increase visualized in green) both in naive (A) and TGF-β-pretreated (50 nM for 24 h; B) permeabilized PGASMC. Top: difference images (increase visualized in purple) and corresponding graphs (red traces) show large mitochondrial [Ca2+] signals closely coupled to the IP3-induced [Ca2+]c rise in naive and only a small increase of [Ca2+]m in TGF-β-pretreated permeabilized cells.

Fig. 5.
Fig. 5.

TGF-β-induced downregulation of type 1 and 3 IP3Rs in PGASMC. Cells were treated with TGF-β1 (50 nM) or vehicle for indicated periods before harvesting. Protein was resolved on a 10% SDS-PAGE and immunoblotted with antibody to IP3R1, IP3R3, and b-actin.

Fig. 6.
Fig. 6.

Two-photon imaging of the NAD(P)H in permeabilized naive and TGF-β-pretreated (50 nM for 24 h) PGASMC. The gray images show the NAD(P)H fluorescence in intact naive (A) and TGF-β-pretreated PGASMC (B) before and after the treatment (5 min) with an uncoupler, FCCP (5 μg/ml, right). The uncoupler was used to stimulate mitochondrial oxidation.

Fig. 7.
Fig. 7.

ER-mitochondrial ultrastructure in naive and TGF-β-treated (50 nM for 24 h) PGASMC. A: micrograph of naive and TGF-β-pretreated PGASMC. B: dimensions of the ER-mitochondrial interface. The average ER-mitochondrial distance (rough and smooth ER to OMM, surface to surface; top) and interface length (with ≤100-nm gap distance) determined from the electron micrographs of PGASMC (150 associations for each condition).

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