**8. Extra-mitochondrial upstream signals that induce TH-induced PTP gating mediated by Bcl2-family proteins**

Since Bcl2-Bax hetrodimerization may depend on Bcl2(S70) phosphorylation state (112), mitochondrial Bcl2 depletion by T3 was further verified in terms of Bcl2(S70) phosphorylation profile. Indeed, concomitantly with decrease in mitochondrial Bcl2, T3 treatment results in decreased phosphorylation of monomeric mitochondrial Bcl2(S70) as well as of Bcl2(S70)-Bax heterodimer (113), indicating that mitochondrial Bcl2 depletion may reflect Bcl2(S70) dephosphorylation by TH. In pursuing kinases (e.g. PKA, PKC) or phosphatases (e.g. PP2A, PP2B/Calcineurin) reported to be involved in Bcl2(S70) phosphorylation (112, 114), neither PKA, PKC nor PP2A were found to mediate phosphorylation/dephosphorylation of Bcl2(S70) by TH (113). In contrast, dephosphorylation of Bcl2(S70) and the depletion of mitochondrial Bcl2 protein by T3 are both reversed by the FK506 inhibitor of PP2B, indicating that the TH effect may be mediated by activation of PP2B (113). Furthermore, added FK506 blocksT3-induced opening of PTP, indicating that dephosphorylation of Bcl2(S70) and its mitochondrial depletion by T3-activated PP2B may account for mitochondrial PTP opening by TH. Since TH-induced PP2B activation was not accompanied by increase in PP2B expression, PP2B activation was further pursued by searching for TH-induced increase in cytosolic Ca+2 (113). Indeed, Ca+2 activated PP2B has previously been reported to bind and dephosphorylate Bcl2(S70) (115, 116). Most importantly, T3 treatment resulted in pronounced increase in Ca+2, while Ca+2 chelation by BAPTA resulted in abrogating LC-PTP gating by TH, indicating that THinduced PP2B activity involved mobilization of intracellular Ca+2 (113). Indeed, T3-induced mobilization of intracellular Ca+2 has recently been reported to mediate a variety of nongenomic effects of TH (117, 118).

The dynamic equilibrium between cytosolic Ca+2 ([Ca+2]c) and endoplasmic reticulum (ER) Ca+2 ([Ca+2]ER) is maintained by an interplay between the inositol 1,4,5-trisphosphate receptor (IP3R1) and the sarcoplasmic Ca+2 ATPase (SERCA) that catalyzes ER Ca+2 efflux and influx, respectively (119). IP3R1 is activated by binding of the IP3 ligand and may further be modulated by its phosphorylation by PKA, PKC or CaMKII, its dephosphorylation by PP2B, or by its association with one or more of about 50 proteins, including FKBP12 or Bcl2 (120, 121). The putative role played by IP3R1 in TH-induced PTP gating was evaluated by verifying the effect of TH in cells lacking IP3R1 (113). Thus, PTP opening, dephosphorylation of mitochondrial Bcl2(S70) and depletion of mitochondrial Bcl2 are all abrogated in cells lacking IP3R1, indicating that IP3R1 is indeed required for THinduced mitochondrial uncoupling. Similarly, T3 is ineffective in increasing [Ca+2]c upon inhibition of IP3R1 by 2APB, indicating a specific requirement for IP3R1 activity in modulating [Ca+2]c by TH. Furthermore, T3-induced gating of IP3R1 is accounted for by both, increase in IP3R1 expression and protein levels, complemented by IP3R1 truncation into channel-only isoforms. Truncated IP3R1 isoforms have been reported to serve as channel-only peptides capable of carrying out [Ca+2]ER efflux in the absence of added IP3 (122-126). IP3R1 truncation by TH may reflect TH activation of IP3R1 proteases that remain to be further verified. The IP3R1 / PP2B crosstalk in mediating TH-induced PTP gating is supported by constitutive PP2B-induced PTP gating under conditions of suppressing IP3R1 expression by siRNA (127). Hence, PP2B is acting downstream to TH-induced IP3R1, and is obligatory as well as sufficient in mediating PTP by [Ca+2]c.

Over all, TH-induced expression of the IP3R1 channel accompanied by its truncation is proposed to result in [Ca+2]ER efflux, increase in [Ca+2]c and [Ca+2]c-activated PP2B, followed by dephosphorylation of mitochondrial Bcl2(S70) with concomitant decrease in mitochondrial Bcl2 protein levels and increase in mitochondrial free Bax (Scheme 1). The decrease in mitochondrial Bcl2 and/or the respective increase in mitochondrial free Bax may initiate and promote variable PTP gating, resulting in physiological LC-PTP– induced calorigenesis. LC-PTP gating may drift to HC-PTP–induced apoptosis as function of additional prevailing conditions that may affect mitochondrial permeability transition.
