Non-canonical hemoglobin: An updated review on its ubiquitous expression

Abstract

Hemoglobin, once thought to be exclusive to erythrocytes, has been identified to be expressed in various cell types over the past several decades. While hemoglobin's function within erythrocytes is primarily characterized as a gaseous transport molecule, its function within non-erythrocyte cells varies among different cell types, and in many cases, remains to be fully elucidated. Despite this variability, hemoglobin expression seems to broadly function as a redox modulator, whether it is involved in the hypoxic response, mitochondrial function, antioxidant balance or, like in erythrocytes, gas transport. This review provides an updated summary of the most recent discoveries of hemoglobin in non-erythrocyte cells. While discussing the function and regulation of this ubiquitous protein, we additionally compare these cell-specific details to identify commonalities throughout the diverse group of hemoglobin-expressing cells. Lastly, we discuss potential implications of non-canonical hemoglobin in various disease states such neurodegeneration, autoimmune disorders, psychological trauma, and hemoglobinopathies, while providing future directions for hemoglobin research.

Highlights

• •Hemoglobin subunits are expressed in a diverse group of cells throughout the body.
• •Non-erythrocyte hemoglobin is hypothesized to modulate various redox reactions.
• •Transcriptional regulation of hemoglobin depends on specific cell type and stimulus.
• •Loss of hemoglobin may influence likelihood or severity of many diseases.

1. Introduction

Adult hemoglobin, the tetrameric protein consisting of both alpha (α) and beta (β) subunits, is canonically known to be expressed in erythrocytes (i.e., red blood cells), where it primarily functions to deliver and exchange gaseous oxygen (O₂) and carbon dioxide (CO₂) throughout the body. While this well-characterized function of hemoglobin is vital for mammalian life, research over the past 30 years has identified this once-thought exclusive erythrocyte protein to be expressed in a myriad of cell types, such as macrophages [1], alveolar epithelial cells [2], mesangial cells [3], retinal pigment cells [4], neurons [5,6], and others. This growing repository of non-canonical cells expressing subunits of hemoglobin was excellently reviewed over a decade ago [7], but numerous reports have emerged since this time demonstrating the highly promiscuous expression of hemoglobin in previously unexplored cell types. In addition to expression, the function and regulation of hemoglobin in previously defined cell types has also begun to be elucidated.

Herein, we discuss the recent updates regarding hemoglobin expression, function, and regulation in various non-canonical cell types. Additionally, we consider how this ubiquitous expression may play a role in several diseases, thus emphasizing the need for further characterization of hemoglobin in these various cell types.

2. Hemoglobin discoveries

2.1. Vascular endothelial cells

One of the cell types with the most well-defined role for non-canonical hemoglobin expression is the vascular endothelial cell. Hemoglobin was initially observed to be expressed in arterial endothelial cells in 2012 [8]. While studying nitric oxide (NO) transport across the junction connecting vascular endothelial cells with smooth muscle cells (i.e., myoendothelial junction), proteomics analysis revealed the presence of high levels of hemoglobin α (Hbα), but no hemoglobin β (Hbβ). Knocking down Hbα in thoracodorsal arteries using siRNA revealed that the loss of Hbα increased NO diffusion across the artery leading to increased vasodilation. The conclusion from this work was that Hbα directly interacts with NO to regulate its diffusion to smooth muscle cells, as opposed to freely diffusing once generated by endothelial nitric oxide synthase (eNOS). Indeed, Hbα was confirmed to be closely associated with eNOS, thus acting as an intermediary for NO once generated. Additionally, the oxidation state of the heme group in Hbα was shown to alter the rate in which NO was released. This observation led to the additional discovery that intracellular cytochrome B5 reductase 3 possessed the ability to reduce the heme iron in Hbα, thus creating a cycle of oxidation/reduction mediated NO sequestration and release [8,9]. Most recently, it was discovered Hbα also functions to reduce nitrite to form NO in vascular endothelial cells, indicating that Hbα also plays a direct role in NO generation in hypoxic arteries, rather than playing a strictly passive role in NO release [10].

The observation that only Hbα was present in vascular endothelial cells was intriguing given that Hbα by itself is unstable, and requires a stabilizing protein such as Hbβ. Due to the lack of Hbβ reported in vascular endothelial cells, another protein, alpha hemoglobin stabilization protein (AHSP) was discovered to bind to endothelial Hbα. The necessity of AHSP for Hbα stabilization in endothelial cells was demonstrated by genetic disruption of AHSP, which led to a decrease in Hbα expression and subsequent dysregulation of NO-mediated vasodilation [9]. However, this is only half of the story. Aforementioned, Hbα is also closely associated with eNOS in vascular endothelial cells, which seemingly also plays a role in both stabilization and reduction of Hbα. In brief, evidence suggests the presence of a redox cycle in vascular endothelial cells where the oxidized iron form of Hbα is stabilized and bound to AHSP, but this AHSP-bound version becomes highly susceptible to iron reduction by eNOS [9]. Once the Hbα iron is reduced by eNOS, it is released from AHSP and stabilized by eNOS where it leads to NO degradation, which oxidizes the iron of Hbα, and once again stabilized and bound by AHSP for another cycle. This elegant cycle allows for finer control of NO diffusion in the vasculature, and demonstrates the malleability of hemoglobin function even in the absence of other subunits [9,11]. With this, the observation of non-canonical hemoglobin not being in the classical tetrameric α-β form is evident throughout many cell types as we will discuss, and demonstrates the dynamic nature of this protein and its potential functions.

While the function of non-canonical hemoglobin in various cell types is often unknown, the genetic regulation of this protein has been another area of research that lacks a clear answer. Due to its considerable history of investigation in red blood cells (i.e., erythrocytes), the regulation of the hemoglobin within erythroid progenitor cells has been extensively characterized, defining transcription factors such as Krupple-like factors (KLF) and GATA [12] to play an important role in its control. However, due to the unique genetic regulation of erythrocytes and the fact that these cells lack a nucleus upon maturation, it is currently unknown if hemoglobin in non-erythroid cells is regulated by similar mechanisms. KLFs are a group of transcription factors that are known to regulate many developmental processes such as cell proliferation, cellular regeneration, hematopoiesis, and endothelial function [13,14]. There is a plethora of research describing the involvement of various KLF isoforms in the regulation of heme synthesis, hemoglobin transcription, and globin switching within erythroid progenitor cells [[15], [16], [17], [18], [19], [20], [21]]. Thus, it was parsimoniously hypothesized that Hbα may be under the control of KLF regulation in endothelial cells as well. Using both endothelial cell-specific knockouts and gain of function KLF2 and KLF4 vectors, Hbα expression respectively responded to both KLF2 and KLF4 manipulation [22]. KLF4 was also identified to bind directly within 300 base pairs upstream of the transcriptional start site of Hbα in endothelial cells, indicating Hbα in endothelial cells is under similar transcriptional regulation as in erythrocytes.

Overall, this unique mechanism of NO interaction and diffusion by endothelial Hbα contributes to minute and rapid changes in vascular tone, which can ultimately affect blood pressure physiology and may have therapeutic potential. To translate these observations into a potential clinical utility, Straub et al. created a Hbα mimetic to disrupt the colocalization of Hbα with eNOS to prevent NO sequestration, thus leading to increased vasodilation [23]. Injection with the Hbα mimetic lowered systolic, diastolic, and mean arterial blood pressure at baseline, and reversed angiotensin II induced hypertension [23]. Additionally, patients with pulmonary hypertension (PH) have elevated levels of Hbα expression in vascular endothelial cells compared to healthy controls [24]. Thus, it was hypothesized that the same Hbα mimetic may be a novel therapy for treating PH. To test this, pulmonary arteries isolated from a mouse model of PH were treated with the Hbα mimetic, which restored the endothelial-dependent dilation response to acetylcholine [24]. Thus, future therapies targeting the eNOS-Hbα colocalization in the endothelial cell vasculature may prove a novel avenue for treating pulmonary (or systemic) hypertension in patients.

2.2. Epithelial cells

Epithelial cells from various organs have also been shown to express hemoglobin. For example, lung epithelial cells were first discovered to express both Hbα and Hbβ approximately 20 years ago [2,25], which was found to be upregulated during times of hypoxia, but its underlying cellular function in these cells remained unknown [26]. Several years later, lung epithelial Hbβ was shown to be involved in NO metabolism and also colocalized with eNOS similar to vascular endothelial cells [27]. However, unlike vascular endothelial cells, interaction of NO with Hbα and Hbβ was identified to provide protection against s-nitrosylation deactivation of soluble guanylate cyclase (sGC) in smooth muscle cells [28]. The deactivation of sGC leads to impaired relaxation of the lungs, which is attributed to airway diseases such as asthma or chronic obstructive pulmonary disease. As such, it was observed in a mouse model of allergic asthma that lung epithelial Hbα and Hbβ was heme deprived, leading to higher levels of NO that were unable to interact with hemoglobin, and thus exacerbated the disease phenotype [28].

Other epithelial cells have also been reported to express hemoglobin, such as vaginal epithelial cells [29], but not all epithelial cells are created equally. While the presence of vaginal hemoglobin was originally believed to originate in the bacterial flora, the expression of both Hbα and Hbβ was later confirmed from human primary vaginal epithelial cells (hPVECs), and were upregulated in response to microbial lipopolysaccharide (LPS) [30]. Immunofluorescence revealed Hbα and Hbβ protein localized to the cytoplasm in hPVECs but was not further analyzed for colocalization with any cytosolic organelle. It was hypothesized that nuclear factor-kappa B (NF-κB), a transcription factor upregulated in response to LPS treatment, may mediate the upregulation of hemoglobin within hPVECs. Indeed, blocking NF-κB did attenuate the characteristic increase of Hbα and Hbβ in response to LPS treatment [30], suggesting a potential link in this factor and the regulation of both hemoglobin subunits. Further analysis via chromatin immunoprecipitation (ChIP) confirmed the interaction of NF-κB with the Hbα promoter, further supporting the role of NF-κB mediated transcriptional regulation. Shortly thereafter, the same group demonstrated the role of nuclear factor erythroid 2-related factor 2 (NRF2) in Hbα and Hbβ expression in hPVECs [31]. Cultured hPVECs treated with hydrogen peroxide (H₂O₂) significantly induced Hbα and Hbβ expression, while cells treated with the NRF2 inhibitor, trigonelline, effectively prevented the increase in both Hbα and Hbβ [31]. Additionally, overexpression of both Hbα and Hbβ significantly decreased reactive oxygen species (ROS) levels in hPVECs, suggesting an antioxidant role for both subunits. Altogether, hemoglobin appears to play a significantly different role within hPVECs compared to its function in lung epithelia or vascular endothelial cells, which also suggests its genetic regulation may be multifaceted and regulated by several factors.

Last, hemoglobin expression has also been observed in syncytiotrophoblasts, or the epithelial cells of the placenta [32]. By examining tissues from healthy and pre-eclamptic (PE) pregnancies, which the latter has been shown to induce hypoxia [33], Hbα protein was identified to be significantly upregulated in syncytiotrophoblasts of PE patients without a significant upregulation of erythrocytes or erythropoiesis. Further, placenta samples from healthy patients cultured in normo- (21 %) and anoxic (0 %) conditions showed robust increases in Hbα mRNA and protein expression in both culture conditions, albeit higher Hbα induction in the anoxic environment, suggesting an O₂-sensing expression response for Hbα in placental cells. Lastly, two potential Hbα transcription factors, hypoxia inducible factor 1α (HIF-1α) and NRF2 were examined, but while both were significantly upregulated in both PE patients and hypoxic placenta samples, both transcription factors failed to localize to syncytiotrophoblasts, thus the authors concluded that neither factor is a likely regulatory candidate in this specific cell type.

2.3. Chondrocytes

One of the most recent observations of non-canonical hemoglobin came in 2023, where Zhang et al. discovered the formation of cytoplasmic, eosin-positive structures within mouse and human chondrocytes [34]. Mass spectrometric analysis revealed these blob-like structures (which they call ‘Hedy structures’) were condensed Hbα and Hbβ subunits, mainly composed of the latter. They discovered that Hedy structures, much like erythrocyte hemoglobin, functions to store O₂ and that the loss of Hbβ results in chondrocytes cell death via hypoxia. In addition to characterizing its function, the group also defined both subunits' expression to be under the transcriptional control of well-known erythrocyte transcription factor Krupple Like Factor 1 (KLF1), which is also known to be upregulated in hypoxia. Overall, the group identified yet another functional role for hemoglobin showing that these unique structures in chondrocytes are transcriptionally-regulated O₂ storage units that are indispensable for mature chondrocyte viability.

2.4. Cells of the nervous system

The expression of hemoglobin within the nervous system is quite extensive, and probably the most well-studied to date. Researchers have identified that various regions of the brain express hemoglobin, such as the hippocampus [[35], [36], [37]] and prefrontal cortex [38,39], as well as a multitude of cell types, such as neurons [5,6,40,41] and astrocytes [5,42,43]. The first record of neuronal hemoglobin expression was published in 1994 in a short communications paper using embryonic whole-brain lysates screened for a set of cDNA probes, where Hbα was identified [44]. Following this, northern blots for both Hbα and Hbβ using primary neuronal cultures confirmed the cell-specific expression of both subunits, which led to a rich area of neuronal hemoglobin research. Since then, several reviews have meticulously outlined the most up-to-date function of hemoglobin within each specific region, developmental period, and disease state of the brain [7,45]. Thus, only the most recent findings will be discussed herein.

Building off previous work suggesting hemoglobin subunits function to store and transport O₂ in neurons [5,40], and that many age-related neurodegenerative diseases such as Alzheimer's have remarkably lower levels of neuronal hemoglobin [39,42,46,47], Lu et al. questioned whether there was an association between aging, hypoxia, and neuronal hemoglobin levels in adult mice [48]. Indeed, a marked decrease in Hbα production was found in the hippocampus and the cerebral cortex of both 18-month-old male and female mice compared to 6- and 12-month-old mice. To further explore the mechanistic function of Hbα within the hippocampus, Hbα was knocked down using a stereotactic injection of a Hbα CRISPR-Cas9 interference lentiviral vector. Hbα knockdown within hippocampal regions resulted in significant HIF-1α expression, indicating the loss of Hbα induced hypoxic conditions. These results suggested that Hbα in the hippocampus at least in part functions to store O₂, as seen in primary cultured neurons [49] and even in glioblastoma cells [50]. Additionally, neuron degeneration was significantly increased after knockdown in the hippocampus and the cerebral cortex [48]. While the exact mechanism in which neuronal hemoglobin expression is decreased in age-related diseases is currently unknown, this work contributes to the growing hypothesis that neuronal hemoglobin functions to protect the brain against hypoxia, and that the loss of this vital protein potentially exacerbates the progression of neurodegenerative diseases.

2.5. Cardiac cells

Hemoglobin has also been discovered to be expressed in multiple cell types of the heart, which has primarily been observed in patients with primary mitral valve regurgitation (PMR) [51]. Originally discovered to be elevated in the pericardial fluid of PMR patients, both Hbα and Hbβ subunits were further identified to be elevated at the mRNA level throughout the chambers of the heart of donor PMR patient hearts compared to healthy controls. While Hbα expression was identified in both cardiomyocytes and interstitial cells via in-situ hybridization (Hbβ in-situ hybridization was not examined), both Hbα and Hbβ protein were only observed in the interstitial cells of PMR hearts, though subcellular localization was not identified. The interstitial cells of the heart are a unique, heterogenous group of cells that help maintain the structure of the heart [52,53], but it was noted that the protein did not localize to cardiac-resident macrophages within the interstitial cells; an essential observation since hemoglobin expression in macrophages has been formerly observed [1].

PMR has been previously characterized to increase cardiomyocyte oxidative stress, mitochondrial damage, and inflammation. Indeed, pericardial fluid from patients with PMR has been shown to exhibit higher levels of proinflammatory cytokines (e.g., IL-6, IL-2, TNFα) and chemokines compared to the patient's circulating plasma, suggesting the activation and recruitment of various immune cells to the cardiac interstitium [51]. While this study served as the initial observation of the expression of hemoglobin protein in interstitial cells of PMR hearts, the authors hypothesize this upregulation of hemoglobin subunits may be a response to oxidative stress. Similar to other non-canonical hemoglobin work, hypoxia was originally considered as a potential stimulus for the upregulation of hemoglobin in PMR hearts. However, hypoxia was ultimately discounted as the primary stimulus due to the lack of HIF-1α induction or myoglobin expression. In contrast, the increase in oxidative stress ‘fossils’ [54] and pro-inflammatory cytokine proteins was suggested as a potential correlative stimulus for hemoglobin expression in cardiac interstitial cells. Ultimately, future work is needed to confirm the cellular source of hemoglobin within the cardiac interstitium, and discern the function of hemoglobin subunits in PMR.

2.6. Immune cells

One of the oldest reports of non-canonical hemoglobin was the observation of Hbβ protein in LPS-stimulated macrophages, where it was hypothesized to function as a potential O₂ or NO sensor [1]. Macrophage hemoglobin was also later seen in a model of sciatic nerve injury, where it was demonstrated that the significant upregulation of both Hbα and Hbβ seen within the damaged tissue was likely coming from a mixture of macrophages and hematopoietic stem cells which had trafficked to the site of injury. [55]. Years later, it was reported that peripheral blood mononuclear cells (PBMCs), a heterogenous group of cells consisting of monocytes, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes, increase hemoglobin protein and mRNA concentration in response to critical illness (i.e., third-degree burns and sepsis) [56]. This upregulation of mainly Hbα subunits, but also Hbβ and Hbδ, was discovered to be from various sources, including hematopoietic progenitor cells present in the patient's blood and increased extracellular hemoglobin from hemolyzed red blood cells subsequently endocytosed by PBMCs. However, the direct expression of hemoglobin subunits by the distinct cell types of PBMCs was not explored in this study. Additionally, the authors also observed endocytosed Hbα colocalized with complex I of the mitochondrial electron transport chain, where it did not alter mitochondrial size or ROS production, but rather increased mitochondrial membrane potential and aspects of mitochondrial metabolism. While this potential mitochondrial role was not further explored within this study, it was hypothesized that the presence of Hbα may function to prevent damage associated with potentially toxic species such as hydrogen sulfide (H₂S), hydrogen peroxide (H₂O₂), peroxynitrite (ONOO⁻), and NO. Indeed, it was demonstrated that incubation of PBMCs with Hbα significantly attenuated cellular damage in response to these species [56], thus providing a cytoprotective function in PBMCs.

Our lab recently reported that T-lymphocytes also express Hbα and Hbβ at both the mRNA and protein level, and thus, may be one of the sources of Hbα in PBMCs as discussed above [57]. Interestingly, only Hbα expression appears to increase in response to ROS (Hbβ, while present, appears differentially regulated), shown by various murine in-vivo ROS-inducing stimuli such as repeated social defeat stress, manganese superoxide dismutase knock-out mice, LPS challenge, as well as many in-vitro redox perturbations, such as H₂O₂ administration, Auranofin supplementation, and hypoxia induction [[57], [58], [59]]. Importantly, Hbα expression and redox induction at the mRNA and protein level was also demonstrated in human T-lymphocytes as well [57], suggesting a conserved mechanism. As previously discussed, others have similarly shown perturbations in the redox environment induce hemoglobin subunits within other non-erythrocyte cells, suggesting an antioxidant-like role of this protein across many cell types. Additionally, we also report an increase in mitochondrial membrane potential and an increase in aspects of mitochondrial metabolism with the overexpression of Hbα, reflecting similar mitochondrial observations as in PBMCs mentioned above [56]. However, we have observed an interesting phenomenon that appears to be unique to T-lymphocytes. Upon activation, Hbα is rapidly downregulated and becomes insensitive to upregulation via ROS, which we demonstrate appears to be via chromatin remodeling [57]. Fascinatingly, this downregulation only occurs in specific subtypes of activated T-lymphocytes (e.g., T_H0, T_H1, T_H2, T_H17), whereas others upregulate Hbα (e.g., T_reg, T_mem), suggesting Hbα expression may play an important role during T-lymphocyte activation and polarization. Interestingly, this downregulatory effect is seemingly opposite in macrophages, where unstimulated macrophages do not produce hemoglobin subunits, but upon stimulation with LPS, the activated macrophages quickly increase hemoglobin mRNA expression [1]. While we have yet to elucidate the details of this complex downregulatory mechanism, we suggest that it is likely due to activation-induced epigenetic silencing and that hemoglobin may act as a sort of activation checkpoint. Future work investigating this complex regulatory mechanism may further our understanding of checkpoints during T-lymphocyte activation which prevent aberrant activation, and may help us understanding the impact on immune function in patients possessing mutant hemoglobin subunits.

3. Hemoglobin in disease phenotypes

In many cases in which hemoglobin has been identified in non-erythrocyte cellular locations, it has been a serendipitous observation while studying various diseases. For example, Hbα and Hbβ expression was first discovered in human hepatocytes while studying nonalcoholic fatty liver disease [60], in the lens cells of the eye studying age related expression changes [61], in mesangial cells of hypoxic kidneys [3], and in T-lymphocytes while examining immune cell gene expression changes after psychological trauma [57]. Additionally, in almost every instance of its discovery in non-erythrocyte locations, hemoglobin seems to generally have a protective role in mediating redox reactions. It is often upregulated in response to an increase in reactive species production or has been identified as significantly downregulated compared to healthy patients in other diseases. Thus, the potential to make hemoglobin part of a biomarker panel in many disease states is feasible, especially in diseases which lack robust biomarkers currently, such as psychological disorders [62]. In fact, there has been a plethora of literature examining the role of hemoglobin in blood pressure regulation [63], Alzheimer's disease [45], Parkinson's disease [47,[64], [65], [66]], multiple sclerosis [[67], [68], [69]], and even implicated in psychological disorders [57,70] (see Table 1). Alternatively, for diseases which directly affect healthy hemoglobin synthesis (i.e., hemoglobinopathies), it was once a mystery why these patients developed seemingly unconnected comorbid diseases. Knowing now that the loss of hemoglobin can negatively affect a myriad of cell types, it is no longer a surprise that losing one or more subunits of hemoglobin coincides with a higher risk for autoimmune diseases [71,72], diabetes [73], stroke [74] and others. One intriguing example of this comes from patients with alpha thalassemia and sickle trait. While these patients indeed suffer from atypical erythrocyte development and O₂ delivery capabilities, the abnormal erythrocytes protect these individuals from severe malaria infection. In contrast, these patients also show vascular dysfunction, and a recently clinical study demonstrated that patients with either alpha thalassemia or sickle trait hemoglobinopathies possess vascular dysfunction due to inappropriate NO regulation by the respective hemoglobin mutations in the vascular endothelium (as opposed to secondary due to erythrocyte deformities) [63]. Targeting the endothelial hemoglobin specifically may provide these patients a possible therapeutic approach to alleviate their vascular dysfunction, while at the same time continuing their inherent protection from malaria due to their erythrocyte hemoglobin mutations. Thus, this provides a prime example of how we should shift our focus into understanding how the loss of hemoglobin within specific non-erythrocyte cell types contributes to the development of these diseases and co-morbidities moving forward.

Table 1.

Overview of hemoglobin discoveries in non-canonical cell types.

Initial Year of Discovery	Cell/Organ Category	Specific Cell Types	Subunits Discovered	Hypothesized Function	Disease Implication	Sources
1994	Brain	Neurons, cells of the hippocampus, glial cells, oligodendrocytes, astrocytes	Hbα-a1, Hbα-a2, Hbβ-b1, Hbβ-b2, Beta-S, Hbγ, Hbδ, Hbζ, Hbε, HbA tetramer	O2 homeostasis, Iron utilization, mitochondrial antioxidant, hemorphins, mitochondrial antioxidant, protection in hypoxia, protection against oxidative stress, epigenetic remodeling, inflammatory response, aids in apoptosis	Alzheimer's disease, Parkinson's disease, multiple sclerosis, prion disease, neurodegeneration, Creutzfeldt-Jakob disease, glioblastoma	[5,6,[35], [36], [37], [38], [39], [40], [41],43,44,[46], [47], [48], [49], [50],66,67,69,75]
1999	Immune	Macrophages, bone marrow mononuclear cells, peripheral blood mononuclear cells, T-lymphocytes	Hbα-a1, Hbα-a2, Hbβ, Hbδ	O2 or NO homeostasis, mitochondrial function, mitochondrial antioxidant	Autoimmune diseases	[1,[55], [56], [57]]
2003	Retinal	Lens cells, retinal pigment epithelium, retina cells, retinal ganglion cells, optic nerve	Hbα-a1, Hbβ-b1, Hbβ-b2, Hbβ-γ, Hbα-X	Iron homeostasis, stress response, O2 homeostasis, protection in hypoxia	Age-related eye diseases, glaucoma	[4,61,76]
2005	Epithelial	alveolar epithelial cells, vaginal epithelial cells, lung epithelial cells	Hbα, Hbβ	O2 or NO homeostasis, protection against oxidative stress, anti-microbial, protection against oxidative stress, maturation and ciliation of airway epithelium	Sleep apnea, pulmonary edema, Asthma, COPD, cystic fibrosis,	[2,[25], [26], [27], [28], [29], [30], [31]]
2008	Kidney	Mesangial cells	Hbα, Hbβ	Protection against oxidative stress		[3]
2008	Reproductive	Endometrium, cumulus cells, sperm, oocytes, granulosa cells, syncytiotrophoblasts	Hbα-a1, Hbα-a2, Hbβ, Hbδ, Hbγ	Iron homeostasis, heme regulation, O2 or NO homeostasis, antioxidant, aid in sperm motility, protection in hypoxia	Preeclampsia	[32,77,77,78,79,80,81]
2011	Liver	Hepatocytes	Hbα-a1, Hbβ	Protection against oxidative stress	Non-alcoholic steatohepatitis	[60]
2012	Endothelial	Arterial endothelial cells	Hbα	NO homeostasis, nitrite reductase	Pulmonary hypertension, vascular dysfunction	[8,10,22,24,63]
2023	Cardiac	Cardiomyocytes, interstitial cells	Hbα, Hbβ, Hbδ, HBε	Hypoxia response, response to inflammation	Primary mitral regurgitation	[51]
2023	Cartilage	Chondrocytes	Hbα, Hbβ	Protection in hypoxia, O2 homeostasis	Rheumatoid arthritis	[34]

4. Conclusion

While hemoglobin subunits have been identified in even more unexpected cell types over the past decade, the protein's overall function(s) within non-erythroid cells remains somewhat elusive. Some research suggests that it may play a role during hypoxia, functioning as an O₂ depot to supply the mitochondrial electron transport chain with a terminal electron acceptor as hypothesized in alveolar epithelial cells [26], cerebral neurons [49], syncytiotrophoblasts [32], and chondrocytes [34]. In contrast, in ovarian follicle cells, it was suggested that hemoglobin binds O₂ not to supply the electron transport chain, but rather to create a pseudo-hypoxic environment to induce HIF1α-mediated events necessary for cell development [77]. Others demonstrate a role in regulating gaseous NO as shown in vascular endothelial cells [8,11,23], lung epithelium [24,27], and in macrophages [1] (along with O₂ transport), or gaseous H₂S, as suggested in human PBMCs [56]. Many demonstrate an antioxidant-like effect of hemoglobin within various cell types, such as a pseudo-peroxidase effect in hepatocytes [60] and mesangial cells [3], an anti-microbial agent in vaginal epithelial cells [30,31], and a less-characterized antioxidant effect in cardiomyocytes [51], T-lymphocytes [57], and cervical cancer cells [82]. It has also been hypothesized to assist with iron/heme metabolism in endometrial cells [78]. Lastly, intracellular hemoglobin has also been suggested to be tightly linked with mitochondrial function. In A9 dopaminergic neurons, it was identified to be expressed in both the cytosol and nucleus, but hemoglobin overexpression significantly altered gene expression of mitochondrial subunits, particularly those of complex I [5]. Inhibition of complex I using rotenone significantly decreased the expression of Hbα and Hbβ in nigral, striatal, and cortical neurons further suggesting an tight correlation of hemoglobin with complex I function in neurons [6]. Others have observed mitochondria localization of hemoglobin in neurons, specifically in the intermembrane space [47], and that translocation of hemoglobin to other areas of the mitochondria may be related Parkinson's disease [65]. In both human PBMCs [56] and T-lymphocytes [57], incubation with hemoglobin or overexpression of hemoglobin lead to a significant increase in mitochondrial membrane potential, as well as increase aspects of mitochondrial metabolism. It was once again specifically localized to complex I of the mitochondria in PBMCs [56], suggesting hemoglobin in multiple cells types may exhibit specific electron transport chain association. Throughout the literature, multiple hypotheses regarding hemoglobin's function(s) within a specific cell type are theorized, but the general theme remains the same throughout all cell types: redox modulation.

Like the function of hemoglobin, its transcriptional regulation within non-erythroid cells also remains up for debate. The regulation of hemoglobin by KLFs and GATA1 in developing erythrocytes is well-characterized, and is known to be upregulated by erythropoietin (EPO) and hemin administration [57]. As discussed above, since mature red blood cells lack a nucleus, and thus have a unique transcriptional regulatory mechanism, it is unknown whether other cell types would be under the same transcription factors. Some reports suggest that hemoglobin is indeed under the control of KLFs, such as KLF2/4 the endothelial cell vasculature [22], KLF1 in chondrocytes [34], and hypothesized in macrophages [1]. Interestingly, KLF3 has been shown to act as a transcriptional repressor of the Hbα locus in non-erythroid cells, and mouse fibroblasts that lack KLF3 significantly upregulate Hbα expression, suggesting a dynamic role for the KLF family in hemoglobin regulation [83]. Others report a role for GATA1 in the regulation of hemoglobin in alveolar epithelial cells [26], and A9 dopaminergic neurons [26]. Since hypoxia is known to induce hemoglobin in many cell types, the HIF1α/EPO signaling cascade has often been examined. A few reports do conclude a role for HIF1α/EPO in hemoglobin expression, such as in retinal cells [76], neurons of the cerebral cortex, cerebellum, hippocampus, and striatum [40], and it is suggested in other areas of the brain [6]. However, many report that despite hypoxia inducing hemoglobin expression, HIF1α does not seem to be the key transcriptional factor [32,34,51,57]. A few studies suggest a role for NF-κB in the regulation of hemoglobin; as seen by the upregulation of hemoglobin in response to LPS in vaginal epithelial cells [30], and involved in the downregulation and epigenetic silencing in T-lymphocytes [57]. Lastly, due to the redox-sensitivity of hemoglobin demonstrated in numerous cell types, NRF2 has also been a possible candidate for hemoglobin expression. One report in vaginal epithelial cells did suggest NRF2 regulating hemoglobin expression in response to H₂O₂ [30], whereas several other studies indicated no role for NRF2 in hemoglobin expression, as seen in syncytiotrophoblasts [32] and T-lymphocytes [57]. Altogether, the regulation of hemoglobin within non-erythroid cells seems to be as diverse as its cellular potency and may have multiple regulatory mechanisms depending on the cell type, developmental timing, and redox stimulus.

Overall, given its ancient history and high conservation among species, it is not surprising that hemoglobin has evolved numerous cellular functions outside of its established role as an O₂ delivery protein. Indeed, it seems that the roles of hemoglobin within the mammalian lifetime span from pre-zygote, important in both sperm maturation [79] and follicular cell oxygenation [77], to embryo development [84], all the way to being implied in age-degenerative diseases, such as glaucoma [76], Alzheimer's [45], Parkinson's [47,[64], [65], [66]], multiple sclerosis [[67], [68], [69]], and cancer [82,85]. It may affect how our central nervous systems metabolize O₂ [49], how immune cells fight pathogens [1,56,57], and how our eyes see the world [4]. Because of this crucial role throughout the body, studies evaluating the role of hemoglobin subunits within non-canonical cell types should take great caution against using whole-body knockout models, and should consider the potential compensatory mechanisms cells may invoke to ameliorate and prevent damage. As such, unless specifically studying the developmental effect of hemoglobin, inducible and temporal knockouts are the suggested model of use to circumvent both developmental defects and compensatory protein upregulation. Additionally, the duplication of the hemoglobin alpha locus, known as HBA1 and HBA2 in humans (and Hba-a1 and Hba-a2 in mice), are identical protein copies, thus in cell types in which both transcripts are detected, one must consider studying both copies to fully elucidate Hbα function. Of course, if studying non-canonical hemoglobin, preventing erythrocyte contamination should be a top priority to prevent erroneous conclusions. Lastly, since hemoglobin is known to respond to redox perturbations, it should be noted that exogenous redox insults to cells may result in cell death, thus in instances in which hemoglobin does not increase or is shown to decrease in response to these insults, cell viability must be considered before drawing conclusions. Collectively, future research continuing to elucidate the function and regulation of hemoglobin in non-erythroid cells may unveil drug-targetable antioxidative mechanisms, posit new biomarkers in age-related and hypoxia-implicated diseases, and provide a better understanding of how hemoglobinopathies affect the entire body of an individual.

CRediT authorship contribution statement

Emily C. Reed: Writing – review & editing, Writing – original draft, Data curation, Conceptualization. Jacob D. Kim: Writing – review & editing, Writing – original draft, Data curation. Adam J. Case: Writing – review & editing, Writing – original draft, Supervision, Software, Resources, Project administration, Funding acquisition, Data curation, Conceptualization.

Declaration of competing interest

The authors have declared that no conflict of interest exists.

Data availability

No data was used for the research described in the article.