Two gaseous waste products perform what must be the greatest atomic shuffle in life. Oxygen exhaled by plants and other photosynthetic lifeforms power aerobic metabolism in animals like us. With each breath, a little carbon escapes [1], lifted on wings of oxygen. This invisible gas was first dubbed “fixed air,” but we know it better as carbon dioxide (CO₂). Given the centrality of CO₂ to our metabolism, perhaps it is not surprising roles are emerging for this previously dismissed waste product in key metabolic pathways, including those involving cholesterol. In this issue of PLOS Biology [2], Bolshette and colleagues link CO₂ levels to the master transcriptional regulator of cholesterol homeostasis, Sterol Regulatory Element Binding Protein-2 (SREBP2).

Our cholesterol concentrations are exquisitely regulated to ensure levels are sufficient yet not toxic. Multiple layers of regulation occur to achieve cholesterol homeostasis, including coordination of a transcriptional program through the elegant SREBP2/Scap pathway [3]. SREBP2 begins life in the membranes of the endoplasmic reticulum (ER). Insufficient cholesterol is sensed by Scap within the ER membranes to allow transport of SREBP2 to the Golgi for proteolytic activation and subsequent induction of genes involved in cholesterol uptake and biosynthesis (Fig 1). When cholesterol levels are sufficient, SREBP2 remains within the ER, switching off the cholesterogenic transcriptional program.

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Fig 1. A simplified model of how varying CO₂ levels affect SREBP2 activation.

Low CO₂ (1%) results in less cholesterol (yellow structure) in the ER allowing SREBP2 to be transported to the Golgi for proteolytic cleavage to release the active TF. However, at physiological CO₂ levels (5%), transport from the ER to the Golgi is inhibited. One possibility is that low CO₂ disrupts cholesterol trafficking between the ER and PM. Created with BioRender.com.

https://doi.org/10.1371/journal.pbio.3002401.g001

Bolshette and colleagues [2] discovered a reduction in CO₂ results in the activation of SREBP2 through decreased levels of cholesterol in the ER via an unknown mechanism. Firstly, using cultured murine fibroblasts as their main cell model, they identified genes with altered transcription in response to changing CO₂ levels. Notably, genes involved in cholesterol biosynthesis and related processes were largely up-regulated or suppressed in response to low (1%) versus normal (5%) or high (10%) CO₂ exposure, respectively. This effect was largely independent of pH changes known to occur with varying CO₂ concentrations. The up-regulation of the cholesterogenic program by low CO₂, the focus of this study, was generalisable to other cell types (notably liver and muscle), although not to primary adipocytes.

SREBP2 was identified as one of the top transcriptional regulators in response to low CO₂. Indeed, immunoblot analysis showed the cleaved, nuclear form of SREBP2 accumulated within only 2 h of low CO₂ exposure. SREBP2 activates transcription of target genes by binding to sterol regulatory elements (SREs) within promoters of genes. Employing an SRE luciferase reporter assay, Bolshette and colleagues [2] observed increased luminescence over time under low CO₂ conditions in cells expressing a wild-type (but not mutated) SRE reporter. This effect was reversible, luminescence fading away when CO₂ levels returned to 5% from 1%.

The translocation of SREBP2 from the ER to the Golgi prior to entering the nucleus is an essential step in SREBP2 activation. Both knockdown of Scap by siRNA and pharmacological inhibition of ER to Golgi trafficking of SREBP2/Scap (using fatostatin) prevented the SREBP2-mediated responses under low CO₂. SREBP2 activation is largely driven by changes in ER cholesterol levels. Low CO₂ did not change total cell cholesterol levels. However, ER levels were depleted, determined after a rigorous cell fractionation protocol [4]. Consistent with this finding, addition of sterols (cholesterol or an oxysterol) inhibited activation of SREBP2 under low CO₂ conditions.

Another recent study [5] reported low O₂ (hypoxia) shuts down the SREBP2 pathway by promoting the ubiquitination and degradation of this transcription factor (TF). However, here the mechanism is very different since Bolshette and colleagues [2] found low CO₂ did not affect SREBP2 stability, but rather, low CO₂ activates SREBP2 target genes by reducing cholesterol in the ER. Accordingly, Bolshette and colleagues [2] found that further depleting cell cholesterol by using cyclodextrin blunted the effect of low CO₂.

But precisely how CO₂ levels influence ER cholesterol levels remains the big question. The fact total cell cholesterol levels remained unchanged after acutely dropping CO₂ levels, but ER cholesterol levels are decreased, suggests the residual cholesterol is trapped elsewhere, perhaps the plasma membrane (PM) where most cell cholesterol resides [4]. Certainly, the phenotype of reduced ER but unchanged total cell cholesterol is reminiscent of a block in PM to ER cholesterol transport observed by others [4]. One scenario is low CO₂ levels destabilize a transporter [6] shuttling cholesterol between the PM and the ER. Increased CO₂ may lead to a posttranslational modification (carbamylation) of lysine residues [7], with the potential to block ubiquitination sites targeting the transporter for degradation. But of course, there are a myriad of other possibilities. Determining if cholesterol derived from lipoproteins also blocks the effect of low CO₂ would help better define the transport defect, considering lipoprotein-derived cholesterol meanders through the endo-lysosomal pathway.

It is unclear whether coupling of CO₂ to cholesterol synthesis may be an adaptive or maladaptive response. Considering cholesterol impedes CO₂ transport across membranes, perhaps an increase in cholesterol synthesis helps maintain intracellular CO₂ levels [8].

What links CO₂ and cholesterol homeostasis in (patho)physiology? Indeed, it is unclear if CO₂ itself or bicarbonate is the active agent, considering the 2 are in rapid equilibrium [9]. Moreover, the physiological relevance of 1% CO₂ is questionable, considering CO₂ levels in blood normally range from 4% to 6% (estimated from 35 to 45 mmHg [10]). However, the general finding is likely still applicable considering Bolshette and colleagues [2] found the effect of CO₂ was graduated from 1% to 10% CO₂. The idea that the response is tuneable across the physiological and pathophysiological range has implications for diseases associated with low (e.g., early asthma) [9] and high CO₂ (e.g., chronic obstructive pulmonary disease) [11].

To conclude, 2 recent studies have implicated each of the key respiratory gasses in regulating cholesterol homeostasis via SREBP2. Analogous to the tussle between O₂ and CO₂ for hemoglobin [7], these 2 gasses have opposing effects on SREBP2 activation and hence regulation of cholesterol biosynthesis genes. Low O₂ prevents SREBP2 activation [5], whereas low CO₂ increases activation [2]. While the O₂- and CO₂-dependent responses and mechanisms appear completely distinct, the signals may cross talk or compete physiologically. Of note, cholesterol biosynthesis is highly O₂-intensive [12] and one of the few biosynthetic processes that also generates CO₂. So, involvement of these 2 gasses in cholesterol homeostasis is perhaps unsurprising. But understanding how CO₂—a carbon atom lifted on wings of oxygen—works its “magic” to help control cholesterol should inspire the field with a breath of fresh air.