Epigenetic Modulation of Class-Switch DNA Recombination to IgA by miR-146a Through Downregulation of Smad2, Smad3 and Smad4.

IgA is the predominant antibody isotype at intestinal mucosae, where it plays a critical role in homeostasis and provides a first line of immune protection. Dysregulation of IgA production, however, can contribute to immunopathology, particularly in kidneys in which IgA deposition can cause nephropathy. Class-switch DNA recombination (CSR) to IgA is directed by TGF-ß signaling, which activates Smad2 and Smad3. Activated Smad2/Smad3 dimers are recruited together with Smad4 to the IgH α locus Iα promoter to activate germline Iα-Cα transcription, the first step in the unfolding of CSR to IgA. Epigenetic factors, such as non-coding RNAs, particularly microRNAs, have been shown to regulate T cells, dendritic cells and other immune elements, as well as modulate the antibody response, including CSR, in a B cell-intrinsic fashion. Here we showed that the most abundant miRNA in resting B cells, miR-146a targets Smad2, Smad3 and Smad4 mRNA 3'UTRs and keeps CSR to IgA in check in resting B cells. Indeed, enforced miR-146a expression in B cells aborted induction of IgA CSR by decreasing Smad levels. By contrast, upon induction of CSR to IgA, as directed by TGF-ß, B cells downregulated miR-146a, thereby reversing the silencing of Smad2, Smad3 and Smad4, which, once expressed, led to recruitment of Smad2, Smad3 and Smad4 to the Iα promoter for activation of germline Iα-Cα transcription. Deletion of miR-146a in miR-146a-/- mice significantly increased circulating levels of steady state total IgA, but not IgM, IgG or IgE, and heightened the specific IgA antibody response to OVA. In miR-146a-/- mice, the elevated systemic IgA levels were associated with increased IgA+ B cells in intestinal mucosae, increased amounts of fecal free and bacteria-bound IgA as well as kidney IgA deposition, a hallmark of IgA nephropathy. Increased germline Iα-Cα transcription and CSR to IgA in miR-146a-/- B cells in vitro proved that miR-146a-induced Smad2, Smad3 and Smad4 repression is B cell intrinsic. The B cell-intrinsic role of miR-146a in the modulation of CSR to IgA was formally confirmed in vivo by construction and OVA immunization of mixed bone marrow µMT/miR-146a-/- chimeric mice. Thus, by inhibiting Smad2, Smad3 and Smad4 expression, miR-146a plays an important and B cell intrinsic role in modulation of CSR to IgA and the IgA antibody response.

Lactate Elicits ER-Mitochondrial Mg2+ Dynamics to Integrate Cellular Metabolism.

Mg2+ is the most abundant divalent cation in metazoans and an essential cofactor for ATP, nucleic acids, and countless metabolic enzymes. To understand how the spatio-temporal dynamics of intracellular Mg2+ (iMg2+) are integrated into cellular signaling, we implemented a comprehensive screen to discover regulators of iMg2+ dynamics. Lactate emerged as an activator of rapid release of Mg2+ from endoplasmic reticulum (ER) stores, which facilitates mitochondrial Mg2+ (mMg2+) uptake in multiple cell types. We demonstrate that this process is remarkably temperature sensitive and mediated through intracellular but not extracellular signals. The ER-mitochondrial Mg2+ dynamics is selectively stimulated by L-lactate. Further, we show that lactate-mediated mMg2+ entry is facilitated by Mrs2, and point mutations in the intermembrane space loop limits mMg2+ uptake. Intriguingly, suppression of mMg2+ surge alleviates inflammation-induced multi-organ failure. Together, these findings reveal that lactate mobilizes iMg2+ and links the mMg2+ transport machinery with major metabolic feedback circuits and mitochondrial bioenergetics.

Mitochondrial pyruvate and fatty acid flux modulate MICU1-dependent control of MCU activity.

The tricarboxylic acid (TCA) cycle converts the end products of glycolysis and fatty acid ß-oxidation into the reducing equivalents NADH and FADH2 Although mitochondrial matrix uptake of Ca2+ enhances ATP production, it remains unclear whether deprivation of mitochondrial TCA substrates alters mitochondrial Ca2+ flux. We investigated the effect of TCA cycle substrates on MCU-mediated mitochondrial matrix uptake of Ca2+, mitochondrial bioenergetics, and autophagic flux. Inhibition of glycolysis, mitochondrial pyruvate transport, or mitochondrial fatty acid transport triggered expression of the MCU gatekeeper MICU1 but not the MCU core subunit. Knockdown of mitochondrial pyruvate carrier (MPC) isoforms or expression of the dominant negative mutant MPC1R97W resulted in increased MICU1 protein abundance and inhibition of MCU-mediated mitochondrial matrix uptake of Ca2+ We also found that genetic ablation of MPC1 in hepatocytes and mouse embryonic fibroblasts resulted in reduced resting matrix Ca2+, likely because of increased MICU1 expression, but resulted in changes in mitochondrial morphology. TCA cycle substrate-dependent MICU1 expression was mediated by the transcription factor early growth response 1 (EGR1). Blocking mitochondrial pyruvate or fatty acid flux was linked to increased autophagy marker abundance. These studies reveal a mechanism that controls the MCU-mediated Ca2+ flux machinery and that depends on TCA cycle substrate availability. This mechanism generates a metabolic homeostatic circuit that protects cells from bioenergetic crisis and mitochondrial Ca2+ overload during periods of nutrient stress.

Blockade of MCU-Mediated Ca2+ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation.

Mitochondrial Ca2+ uniporter (MCU)-mediated Ca2+ uptake promotes the buildup of reducing equivalents that fuel oxidative phosphorylation for cellular metabolism. Although MCU modulates mitochondrial bioenergetics, its function in energy homeostasis in vivo remains elusive. Here we demonstrate that deletion of the Mcu gene in mouse liver (MCUΔhep) and in Danio rerio by CRISPR/Cas9 inhibits mitochondrial Ca2+ (mCa2+) uptake, delays cytosolic Ca2+ (cCa2+) clearance, reduces oxidative phosphorylation, and leads to increased lipid accumulation. Elevated hepatic lipids in MCUΔhep were a direct result of extramitochondrial Ca2+-dependent protein phosphatase-4 (PP4) activity, which dephosphorylates AMPK. Loss of AMPK recapitulates hepatic lipid accumulation without changes in MCU-mediated Ca2+ uptake. Furthermore, reconstitution of active AMPK, or PP4 knockdown, enhances lipid clearance in MCUΔhep hepatocytes. Conversely, gain-of-function MCU promotes rapid mCa2+ uptake, decreases PP4 levels, and reduces hepatic lipid accumulation. Thus, our work uncovers an MCU/PP4/AMPK molecular cascade that links Ca2+ dynamics to hepatic lipid metabolism.