Calcium acts as a critical determinant of Mitochondria-Nuclear networking driven retrograde signaling

Abstract

Mitochondria are robust signaling organelle that regulate a variety of cellular functions. One of the key mechanisms that drive mitochondrial signaling is inter-organelle crosstalk. Mitochondria communicates with other organelles primarily via exchange of calcium (Ca²⁺), reactive oxygen species (ROS) and lipids across organelle membranes. Mitochondria has its own genome but a majority of mitochondrial proteins are encoded by nuclear genome. Therefore, several mitochondrial functions are controlled by nucleus via anterograde signaling. However, the role of mitochondria in driving expression of genes encoded by nuclear genome has recently gained attention. Recent studies from independent groups have demonstrated a critical role for mitochondrial Ca²⁺ signaling in stimulating nuclear gene expression. These studies report that inhibition of mitochondrial Ca²⁺ uptake through silencing of Mitochondrial Ca²⁺ Uniporter (MCU) leads to Ca²⁺ oscillations in the cytosol. The rise in cytosolic Ca²⁺ results in activation of Ca²⁺ sensitive transcription factors such as NFATs and NF-κB. These transcription factors consequently induce expression of their target genes in the nuclear genome. It is important to highlight that these groups used different cell types and elegantly presented a phenomenon that is conserved across various systems. Notably, mitochondrial Ca²⁺ signaling mediated transcriptional regulation controls diverse cellular functions ranging from B-cell activation, melanogenesis and aging associated inflammation. Future studies on this signaling module would result in better understanding of this axis in human pathophysiology and could lead to development of novel therapeutic strategies.

Mitochondria, apart from their well-characterized role as cellular bioenergetic factories, perform vital role as intracellular signaling hubs. Mitochondria crosstalk with a variety of organelles to drive cellular physiology and any perturbations in such inter-organelle communication leads to human pathologies [1,2]. One of the key drivers involved in mitochondrial inter-organellar communication is mitochondrial Ca²⁺ signaling. Mitochondrial Ca²⁺ dynamics orchestrate several physiological and pathological functions in the cell by intricate regulation of organellar Ca²⁺ levels [3]. Mitochondrial Ca²⁺ uptake majorly occurs in specialized regions of close proximity between ER and mitochondria, called mitochondria associated membranes (MAMs), which form nanodomains comprising of IP3Rs (localized on ER membrane) and VDACs (localized on outer mitochondrial membrane, OMM). Mitochondrial Ca²⁺ uniporter (MCU), a highly selective multi-subunit channel present on the inner mitochondrial membrane mediates Ca²⁺ uptake into the matrix [4].

Predominantly, signaling modules initiated in the nucleus have been attributed for shaping the cellular architecture and physiology. Nuclear transcription regulates the organellar protein profile either directly or indirectly by post-translational control of events like protein degradation, assembly or enzyme activity [5]. Such a chain of events dictated by the nucleus that modulates organellar function is widely recognized as anterograde signaling. Conversely, in retrograde signaling, organelles such as mitochondria and chloroplasts communicate with the nucleus to drive physiological responses. So far, ROS accumulation, redox state, certain metabolites and transcription factors originating from the organelle have been recognized as retrograde signals [6]. However, the critical contribution of organellar Ca²⁺ signaling, especially the utilization of mitochondrial Ca²⁺ as the currency for retrograde signaling is still an emerging field. The mitochondrial Ca²⁺ handling machinery is accountable for shaping the organellar Ca²⁺ dynamics and recent literature reveals how it can regulate various downstream processes via nuclear gene expression regulation.

In an interesting study, Trebak lab reported that MCU mediated mitochondrial Ca²⁺ uptake tunes the cytosolic and endoplasmic reticulum Ca²⁺ signaling in mammalian cells, which further modulates transcription and downstream cellular functions [7]. Mitochondria, via MCU complex, can buffer Ca²⁺ in response to elevation in cytosolic Ca²⁺ levels. This ensures maintenance of cytosolic Ca²⁺ homeostasis. When MCU is inhibited, the pace of cytosolic Ca²⁺ oscillations and ER Ca²⁺ refilling is enhanced. Experiments done in a broad range of non-excitable mammalian cell lines and primary cells show that MCU depletion is responsible for heightened cytosolic Ca²⁺ signals, ER store refilling and NFAT1/4 nuclear translocation.

However, in this scenario, neither the IP3R levels are affected, nor is their activity. In the absence of MCU, mitochondrial Ca²⁺ uptake declines severely, leading to Ca²⁺ buildup in the cytosol and increased Ca²⁺ uptake by the ER. The Ca²⁺ microdomains in MCU-KO cells induce NFAT1/4 nuclear translocation. Further, experiments performed using mice derived CD4⁺ T cells and B cells with silenced MCU demonstrate that this cascade results in enhanced store operated Ca²⁺ entry (SOCE) in both T and B-cells along with higher B-cell proliferation.

An exciting study from Desai lab demonstrated a crucial role of MCU mediated mitochondrial Ca²⁺ signaling in aging associated inflammation i.e. ‘inflammaging’ [8]. The term ‘inflammaging’ encompasses chronic, low grade inflammatory responses and high levels of aging associated circulating biomarkers such as interleukin-6 and C-reactive protein. Literature suggests that aging is associated with diminished enzymatic activity of key mitochondrial enzymes, decline in mitochondrial respiratory capacity and increase in ROS levels [9]. Further, dysfunctional mitochondria release mtDNA and formyl peptides to activate innate immune system during aging. However, role of mitochondrial Ca²⁺ signaling in molecular aging remained poorly understood. Desai lab carried out transcriptomic analysis on 700 human blood samples and revealed that inflammatory response related genes especially those linked to the NF-κB pathway were higher in the oldest group (age 60-69 years) when compared to the youngest population (age 20-29 years). Further, they observed a concomitant decline in the genes related to mitochondrial function (OXPHOS, Ca²⁺ signaling etc.). Interestingly, a tissue-specific but robust decline in MCU expression was observed in aged samples. The authors demonstrated that MCU silencing mediated reduction in mitochondrial Ca²⁺ uptake in macrophages triggered cytosolic Ca²⁺ oscillations. This consequently induced higher nuclear translocation of NF-κB, which in turn led to the transcription of pro-inflammatory genes (IL-6 and IL-1β). However, the precise mechanism behind the mCa²⁺ mediated NF-κB translocation remains to be explored in detail. MCU deletion in macrophages originating from young mice led to a hyper-inflammatory response upon stimulation, implying the role of reduced mCa²⁺ uptake in inflammation. Taken together, these observations link decreased mitochondrial Ca²⁺ uptake to inflammaging. The convergence of aging and inflammation with mitochondrial Ca²⁺ signaling opens up novel arenas of research targeted towards tackling the aging-related pathologies or slowing down of aging itself.

Recent work from our lab shows the importance of mitochondrial Ca²⁺ signaling in driving vertebrate pigmentation [10]. Using four independent model systems (B16 mouse melanoma cells, primary human melanocytes, zebrafish and transgenic mouse model), we show that MCU mediated mitochondrial Ca²⁺ uptake triggers melanogenesis. MCUb, a dominant negative component of MCU complex, negatively regulates mitochondrial Ca²⁺ uptake and pigmentation. To delineate the molecular mechanisms that connect mitochondrial Ca²⁺ signaling to melanogenesis, we performed unbiased RNA seq on melanocytes with MCU or MCUb silencing and identified an interesting differential regulation of keratin filaments. Further, we show that decrease in mitochondrial Ca²⁺ levels results in activation and nuclear translocation of NFAT2 transcription factor, which in turn induce keratin5 transcription. Keratin5 further regulates pigmentation downstream of mitochondrial Ca²⁺ signaling. In summary, we report a novel MCU-NFAT2-keratin5 mediated signaling axis that plays a critical role in vertebrate pigmentation. Importantly, we show that inhibition of MCU with an FDA approved drug-mitoxantrone decreases physiological pigmentation. This data suggests that mitochondrial Ca²⁺ signaling could be targeted for management of pigmentary disorders.

Taken together, work from independent groups demonstrate that decrease in mitochondrial Ca²⁺ uptake via MCU silencing leads to activation of Ca²⁺ sensitive transcription factors, which in turn induce cell and context dependent gene expression programs. This consequently regulates critical cellular functions and contribute to aging associated inflammation (Figure 1). Further studies on this signaling axis would lead to its detailed characterization and delineating its potential relevance in pathophysiological conditions. Moreover, it would be interesting to investigate if NFATs and NF-κB regulate expression of mitochondrial localized proteins in particular MCU complex proteins thereby generating a feed forward loop for regulating mitochondrial Ca²⁺ signaling.

Figure 1. Mitochondrial Ca²⁺ regulates diverse cellular functions through activation of Ca²⁺ sensitive transcription factors.

A decline in MCU mediated mitochondrial Ca²⁺ import leads to a concomitant increase in the cytosolic Ca²⁺ levels. As a response, Ca²⁺ sensitive transcription factors such as NFAT and NFκB are activated and translocated into the nucleus, where they trigger nuclear gene transcription and induce biological outcomes. For example: NFAT1/4 activation and nuclear translocation potentiates B-cell proliferation, NFAT2 mediated transcriptional changes alter cellular melanogenic profile and NFκB mediates the process of inflammaging.