The Transcription Factor HOXA5: Novel Insights into Metabolic Diseases and Adipose Tissue Dysfunction

The transcription factor HOXA5, from the HOX gene family, has long been studied due to its critical role in physiological activities in normal cells, such as organ development and body patterning, and pathological activities in cancer cells. Nonetheless, recent evidence supports the hypothesis of a role for HOXA5 in metabolic diseases, particularly in obesity and type 2 diabetes (T2D). In line with the current opinion that adipocyte and adipose tissue (AT) dysfunction belong to the group of primary defects in obesity, linking this condition to an increased risk of insulin resistance (IR) and T2D, the HOXA5 gene has been shown to regulate adipocyte function and AT remodeling both in humans and mice. Epigenetics adds complexity to HOXA5 gene regulation in metabolic diseases. Indeed, epigenetic mechanisms, specifically DNA methylation, influence the dynamic HOXA5 expression profile. In human AT, the DNA methylation profile at the HOXA5 gene is associated with hypertrophic obesity and an increased risk of developing T2D. Thus, an inappropriate HOXA5 gene expression may be a mechanism causing or maintaining an impaired AT function in obesity and potentially linking obesity to its associated disorders. In this review, we integrate the current evidence about the involvement of HOXA5 in regulating AT function, as well as its association with the pathogenesis of obesity and T2D. We also summarize the current knowledge on the role of DNA methylation in controlling HOXA5 expression. Moreover, considering the susceptibility of epigenetic changes to reversal through targeted interventions, we discuss the potential therapeutic value of targeting HOXA5 DNA methylation changes in the treatment of metabolic diseases.


Introduction
HOX genes are the largest and most extensively studied members of the major Homeobox transcription factor (TF) superfamily, which plays a significant role in embryonic development, hematopoiesis, and tumorigenesis [1]. The HOX gene family consists of 39 genes clustered in four chromosomal regions (HOXA, HOXB, HOXC, and HOXD). HOX genes encode TFs that bind via their homeodomain DNA motifs in HOX-responsive elements to regulate tissue-specific gene expression and thus direct the morphogenetic events leading to complex body forms [2]. Consequently, any mutations and unregulated expression of the HOX genes can lead to anomalies at various stages of development, causing aberrant phenotypes in humans [3,4].
The transcription factor HOXA5 was initially identified because of its essential role in axial skeleton formation during development [5,6]. Subsequently, the analysis of HOXA5 mutant mice has revealed a plethora of phenotypes, including organ defects and postnatal abnormalities, which are indicative of the broad range of actions of this gene throughout life. Indeed, an aberrant HOXA5 expression contributes to anomalies and dysfunction in various organs, such as the thyroid gland, mammary glands, and ovaries, as well as affecting proliferation, differentiation, invasion, apoptosis, and other biological processes in multiple cancer types [5,6], where HOXA5 can function as both an oncogene and tumor suppressor [7].
Besides this well-described role in normal development and carcinogenesis, increasing evidence further indicates that HOXA5 is highly expressed in the adipose tissue (AT) and plays an active role in regulating adipocyte biology and body fat distribution [8]. Changes in HOXA5 expression correlate with variations in adiposity levels and the pattern of body fat distribution [9]. Furthermore, extreme weight loss in obese humans is associated with the upregulation of the HOXA5 gene in the subcutaneous AT (SAT), which implicates this transcriptional regulator in the expansion of AT [10,11]. The ability to recruit new adipocytes through adipogenesis is critical for a healthy AT expansion and systemic metabolic health in the setting of caloric excess [12]. We demonstrated that HOXA5 is essential for proper adipogenesis in humans and mice [11,13], which would make it an appealing molecular target for the treatment of metabolic pathologies related to AT dysfunction since modulating adipogenesis is now emerging as a viable therapeutic strategy to promote healthy AT remodeling and an improved metabolic state during overnutrition [14].
Epigenetics adds a further layer of complexity to HOXA5 gene regulation in relation to metabolic diseases [15,16]. DNA methylation is the most common strategy for the control of the dynamic HOXA5 expression profile [5,6]. Indeed, an increased methylation in the Hoxa5 promoter region contributes to its downregulation in the AT of mice exposed to a high-fat diet [11]. In addition, increase in methylation at the HOXA5 promoter reduces its expression in the SAT pre-adipocytes of individuals with hypertrophic obesity, a wellestablished condition of restricted adipogenesis [13]. Besides its pathophysiological role, the methylation changes at the HOXA5 locus may also act as biomarkers to predict the risk of obesity and type 2 diabetes (T2D), in line with the current view that methylation changes detected in bloodborne DNA may represent a mark for other tissues more directly implicated in disease pathogenesis [17]. Indeed, blood DNA methylation levels at the HOXA5 gene were increased in subjects with a family history of T2D with hypertrophic obesity, and this was associated with increased adipose cell size, an independent predictor of T2D [13]. In obese individuals, blood DNA methylation at the HOXA5 promoter strongly correlates with body mass index (BMI), indicating that the HOXA5 methylation profile may also indicate the level of adiposity [13,18]. An intriguing feature of epigenetic changes is their dynamic nature and potential reversibility in response to targeted interventions [17], raising the possibility of developing a promising HOXA5-targeted therapeutic strategy.
Given the growing body of evidence linking HOXA5 to the pathogenesis of obesity and T2D, a comprehensive review of its functions and mechanisms in regulating AT function is required. The potential application of HOXA5 DNA methylation changes in the evaluation of disease risk and the efficacy of such an intervention will be discussed. A flowchart of the study selection process is presented in Figure S1.

HOXA5: A Crucial Transcription Factor in Development and Disease
The HOXA5 gene encodes a 270-amino-acid protein with a highly conserved DNAbinding homeodomain, by which HOXA5 finely regulates the mRNA expression of target genes determining cell and tissue identities during organism development [19].
The evidence for the essential role of Hoxa5 in developmental and organogenesis comes mainly from the study of the Hoxa5 knock-out mice [20,21]. While heterozygous Cells 2023, 12, 2090 3 of 14 mutants (Hoxa5 +/− ) were viable and indistinguishable from their wild-type littermates, the viability of homozygous mutants was markedly reduced with 50% of the mutant animals dying at birth or shortly thereafter from respiratory distress due to improper tracheal and lung morphogenesis [21]. Hoxa5 absence leads to a significantly diminished expression of the Nkx2-1 (NK2 homeobox 1) and Foxa2 (Forkhead box A2), known key TFs governing lung epithelial cell differentiation and formation [22]. Survival Hoxa5 −/− mice develop an emphysema-like phenotype characterized by lung airspace enlargement [23]. Several Hoxa5 mutant phenotypes closely resemble the phenotype in human lung pathologies. Consistently, altered HOXA5 expression has been reported in patients with lung developmental diseases and adult pathologies [24][25][26]. These findings support the concept that genetic alterations affecting the HOXA5 gene during early-life organogenesis may trigger an adult onset disease [17,27]. This is in line with recent evidence for the role of developmental genes in the origin of metabolic diseases, including obesity and altered body fat distribution [8,28]. HOXA5 also regulates the function of the gastrointestinal tract. Indeed, its absence perturbs the gastric enzymatic activity and the cell specification of the gastric epithelium, resulting in a delay of intestinal acquisition of an adult-mode digestion [29]. Further, the lack of Hoxa5 function alters thyroid development, as shown by smaller and disorganized follicles, causing transient hypothyroidism [30]. HOXA5 has been shown to play a role in blood differentiation in humans. Indeed, sustained HOXA5 expression in human hematopoietic progenitor cells causes a significant shift towards myeloid differentiation and away from erythroid differentiation [31,32]. Finally, HOXA5 could substantially impact the immune microenvironment via the regulation of Th2 cells (T helper cells, also known as CD4 + cells, are a type of T cell that play an important role in the adaptive immune system by producing a variety of cytokines) and M2 macrophages (defined as alternative activated macrophages which are mainly involved in anti-inflammatory responses). Indeed, Hoxa5 alleviates obesity-induced chronic inflammation by promoting M2 macrophage polarization in mouse AT [15,33,34]. In summary, perturbed cell proliferation, impaired differentiation, and altered remodeling of cell morphogenesis are common features in Hoxa5 mutant mice [6].
Since the aforementioned pathophysiological processes are hallmarks of cancer cells, it is not surprising that deregulated HOXA5 expression is associated with oncogenesis [35]. Nearly 70% of all breast carcinomas have decreased HOXA5 expression levels [36], and its loss correlates with progression to higher-grade lesions. Indeed, in breast cancer, HOXA5 exerts its tumor suppressor function by regulating p53 [37]. Reduced HOXA5 expression is also a biomarker for poor prognostic in human non-small-cell lung cancer (NSCLC) [38]. Its overexpression significantly suppresses invasive capability in lung cancer by reducing the expression levels of several actin remodeling-related genes [39], a finding that is in agreement with the role of HOXA5 in AT remodeling in obesity conditions [11]. Downregulation of the HOXA5 gene in NSCLC can occur following HOXA5 suppression by the microRNA-196a or due to aberrant promoter methylation [26,40]. Altered DNA methylation of HOXA5 has also been linked to gastric tumorigenesis [41]. In colorectal cancer, the extent of HOXA5 methylation is associated with its clinic-pathological characteristics [42]. According to the mainly suppressive nature of DNA methylation, the HOXA5 gene is hyper-methylated and downregulated in colon cancer, and its re-expression induces loss of the cancer stem cell phenotype, preventing tumor progression and metastasis by inhibiting the β-catenin/WNT (Wingless-related integration site) signaling pathway [43]. The same situation has also been found in human pre-adipocytes, where HOXA5 promoted adipogenesis by restraining WNT signaling [13]. Hyper-methylation of HOXA5 and the subsequent reduction of its expression in leukemic cells correlate with a poor prognosis in chronic and acute myeloid leukemia [44,45]. Despite this well-established function as a tumor suppressor, several studies indicate that HOXA5 may act as an oncogene in certain organs. Indeed, HOXA5 expression is increased in gliomas, which is associated with increased tumor aggressiveness [46,47]. Thus, either a gain or a loss of Hoxa5 gene expression can deregulate several signaling pathways, potentially triggering a transcriptional program in the hit tissues, disturbing normal development, and leading to tumorigenesis.

Role of HOXA5 in Metabolically Unhealthy States
AT is now recognized as a highly active metabolic and endocrine organ impacting the whole-body energy homeostasis. However, it remains the primary site for energy storage and utilization. SAT is the largest adipose depot, storing around 80% of total body fat [48]. AT is composed of several cell types, including mature adipocytes, pre-adipocytes, and a range of inflammatory cells. The size of mature adipocytes has been widely used to link cellular and tissue functions to metabolic health. Healthy AT expands through a combination of hypertrophy (the enlargement of mature adipocytes) and hyperplasia (the recruitment and differentiation of pre-adipocytes). In obesity, AT may become severely dysfunctional and not expand properly to store the energy excess due to the inability to recruit new adipocytes through adipocyte differentiation and hypertrophic enlargement of pre-existing adipose cells. Moreover, after SAT has reached its limit for hypertrophic expansion, further caloric overload accumulates in ectopic locations, including key metabolic tissues like the liver and skeletal muscle. It has been largely demonstrated that these abnormalities result in hypertrophic obesity, local inflammation, and IR, increasing the risk of developing T2D [49][50][51].
An abnormal HOXA5 expression in the AT, caused in part by epigenetic dysregulation, may impair normal development and differentiation programs resulting in malfunction at the cellular and tissue levels, which are primary abnormalities in obesity and its related illnesses. The role of HOXA5 in the pathophysiology of metabolically unhealthy states such as AT hypertrophy, obesity, and T2D will be explored below.

HOXA5 and Hypertrophy of AT
Adipose cells develop from fibroblast-like pre-adipocytes to fully differentiated lipidenriched adipocytes [51]. The transcriptional regulatory network controlling the maturation of adipocytes has been the focus of intense research and is reviewed elsewhere [52]. Here we discuss the central role of HOXA5 in this process. HOXA5 is highly expressed in AT [8], with expression induced early in pre-adipocytes, and continues to increase throughout the course of adipocyte differentiation in both humans and mice [11,13,53]. Gain or loss function of Hoxa5 in murine pre-adipocyte cell models promoted or inhibited adipogenesis, respectively [11,54]. As in rodents, in human SAT pre-adipocytes, HOXA5 depletion impaired adipocyte differentiation, which results in reduced lipid accumulation and decreased expression of key adipogenic markers [13].
While the requirement of HOXA5 for adipogenesis is hereby conserved from mouse to human, the mechanisms responsible for its function in this process remain largely unknown. A first relevant contribution to this issue comes from our recent study where we reported that the silencing of HOXA5 in human pre-adipocytes caused dysregulation of the transcriptional program at several genes in the WNT-signaling pathway, ultimately leading to impaired adipogenesis [13]. The pathophysiological relevance of these in vitro results was further supported by the findings that some of these WNT components, including the anti-adipogenic factors NFATC1 (Nuclear factor of activated T cells 1) and WNT2B (Wnt family member 2B), were also significantly upregulated in SAT-derived pre-adipocytes of individuals with hypertrophic obesity, a well-established condition linked to restricted adipogenesis and characterized by low HOXA5 expression [13,55,56]. In these subjects, remarkably, the NFATC1 and WNT2B expression negatively correlated with that of HOXA5, supporting the negative regulation of HOXA5 on the transcriptional activity of NFATC1 and WNT2B. Considering that inappropriate activation of WNT-signaling prevents adipocyte differentiation and associates with hypertrophic adipocyte [57], it is possible that, in human pre-adipocytes, HOXA5 promoted adipogenesis by restraining WNT-signaling, thus leading to healthy AT expansion. These observations are in keeping with in vivo rodent studies, where mice injected subcutaneously with adenovirus-Hoxa5-shRNA (short hairpin RNA) exhibited an AT Cells 2023, 12, 2090 5 of 14 characterized by hypertrophy of adipose cells, whereas Hoxa5-overexpressing mice featured reduced adipose cell size. Remarkably, the overexpression of Hoxa5 resulted in a simultaneous reduction in body and AT weight, and improved insulin sensitivity. Additionally, the elevated expression of Hoxa5 led to a decrease in triglycerides and levels of inflammatory cytokines. Conversely, Hoxa5 silencing had adverse effects on the metabolic profile of the animals [34]. Further research has also indicated that Hoxa5 promotes adipocyte differentiation by transcriptionally activating the key adipogenic factor Fabp4 (Fatty acid binding protein 4) while inhibiting the PKA/HSL (Protein kinase A/Hormone-sensitive lipase) signaling pathway, whose dysregulation results in adipocyte hypertrophy [54,58]. Consistently, reduced Fabp4 expression was detected in multiple Hoxa5 null tissues [59]. In addition, both HOXA5 and FABP4 expression in human SAT were associated with elevated circulating triglycerides levels, which represent a feature of hypertrophic obesity [16,60]. Interestingly, Li Y et al. have demonstrated that Hoxa5 induced the differentiation of pre-adipocytes by directly controlling the expression of microRNA-574-5p, whose downregulation in SAT is associated with adipocyte hypertrophy and IR in vivo [61]. Additionally, during hypertrophic obesity, the expansion of AT results in the activation of apoptotic signaling, including death receptor and mitochondrial pathways [62]. Notably, Feng F et al. have reported that a dysregulated expression of Hoxa5, upon palmitic acid exposure, induced apoptosis in mice adipocytes by altering the transcription activity of the pro-apoptotic gene Bax (BCL2-associated X protein), which is involved in the mitochondrial pathway [63]. These findings further demonstrate the regulatory role of Hoxa5 in adipocyte function.
Despite the strength of these observations, several questions remain. We still have to comprehensively identify the direct targets of HOXA5 that contribute to establishing the regulatory landscape during the promotion of adipocyte differentiation. Our unpublished data from transcriptome analysis of HOXA5-silenced human pre-adipocytes revealed that, during adipocyte differentiation, HOXA5 could modulate a consistent set of genes, representing specific functional categories, including adipocyte metabolism, remodeling of adipocyte morphology, and a number of genes whose function in adipogenesis remains to be unearthed.
Thus, HOXA5 stands out as an essential regulator of adipogenesis-both in vitro and in vivo-making it, along with its gene network, an intriguing molecular target for the treatment of AT dysfunction and associated metabolic diseases.

HOXA5 in Obesity and Type 2 Diabetes
As a crucial regulator of adipose cell development, HOXA5 is required for proper lipid storage in the AT and may thus contribute to obesity. HOXA5 is highly expressed in both visceral and SAT under physiologic conditions. Strikingly, HOXA5 is strongly downregulated in both visceral and SAT from individuals who have obesity, and its expression is inversely associated with both BMI and waist-to-hip ratio (WHR) [8,28,64]. Animal studies, where we and others have demonstrated that the expression of HOXA5 was significantly reduced in the abdominal fat depots of diet-induced and genetically obese mice, provide additional evidence for the role of HOXA5 in the pathophysiology of obesity [8,11,65]. Although AT in visceral fat depot has long been considered a major contributor to the development of metabolic disorders, a growing body of evidence indicates that abdominal SAT depot also exerts a significant impact on the development of IR and T2D. Gene expression analysis of different SAT depots revealed that HOXA5 expression was higher in abdominal SAT than in gluteal subcutaneous depots, and these same differences were observed in SAT fractions, including adipocytes and stromal-vascular fraction (SVF) cells. Thus, HOXA5 expression is related to the degree of central obesity and the pattern of body fat distribution and, by extension, should be associated with an increased risk for obesity-related abnormalities such as IR and T2D. This is supported by the evidence from our recent study on lean subjects who were first-degree relatives of T2 diabetics (FDR), which are at high risk of developing the disorder. These subjects exhibit subcutaneous adipocyte hypertrophy due to restricted adipogenesis of resident pre-adipocytes, which make their abdominal SAT similar to those of obese subjects, even if not obese [66]. The storage capacity of their abdominal SAT is limited, and further caloric overload could promote ectopic fat accumulation, leading to local inflammation and IR fostering the onset of T2D. Remarkably, HOXA5 expression was significantly downregulated in the pre-adipocytes of these subjects, and its reduced mRNA levels negatively correlated with the increased size of their subcutaneous adipocytes [9]. A downregulation of HOXA5 was also observed in isolated adipocytes from the abdominal SAT of obese Pima Indians, a population with one of the highest prevalence rates of obesity and T2D [13,67]. Besides, individuals who have obesity and T2D showed reduced expression of HOXA5 in abdominal SAT [61]. Therefore, the HOXA5 deficit is a distinct feature of a dysfunctional abdominal SAT and may represent a marker of an increased risk of developing unfavorable metabolic outcomes, either in individuals with T2D familiarity or in subjects with obesity.
Changes in HOXA5 expression have also been reported following weight/fat loss interventions. In a study investigating fat depot-specific transcriptome signatures before and after exercise-induced weight loss in obese African women, HOXA5 was one of three genes with higher expression in abdominal vs. gluteal SAT in response to exercise training [68]. Furthermore, profound weight loss following bariatric surgery in obese individuals was accompanied by the upregulation of the HOXA5 gene in the abdominal SAT [10]. Similarly, high-fat diet (HFD)-induced obese mice achieving significant weight loss after caloric restriction treatment revealed increased HOXA5 expression in AT. These studies demonstrate that the expression of HOXA5 is responsive to variations in fat mass upon weight loss interventions, further strengthening the functional relevance of this TF in AT by regulating multiple genes involved in AT remodeling [69]. It is also possible that the HOXA5 transcription factor may control inflammation, such that its decreased AT expression during obesity may release the brake on pro-inflammatory genes, limiting tissue repair capability. A protective role of Hoxa5 was reported in the studies of Cao W et al., which demonstrated that Hoxa5 alleviates obesity-induced chronic inflammation by reducing pro-inflammatory cascades, including ER stress-and TNC/TRL4/NF-κB (Tenascin-C/Toll-Like Receptor 4/Nuclear Factor Kappa B) inflammatory pathways and, importantly, by promoting anti-inflammatory M2 macrophage polarization in mouse AT [34,70]. Similarly, forced Hoxa5 expression attenuated carotid atherosclerosis-related inflammation in mice through enhancing macrophages switching toward the anti-inflammatory M2 phenotypes. In this context, Hoxa5 exerted an anti-inflammatory function by regulating the Med1 (Mediator complex subunit 1) gene [71], which also acts as a lipogenesis coactivator required for proper AT expansion [72]. These findings suggest that HOXA5 promotes metabolically favorable AT remodelling in response to changes in body fat composition by suppressing inflammatory cascades while upregulating anti-inflammatory signals both in vitro and in vivo. The lungs of Hoxa5 null mice consistently featured a perpetuated infiltration of activated macrophages [33,73]. Inflammatory pathways are being targeted as a novel treatment method to improve metabolic health [74]. Therefore, Hoxa5 may be a potential molecular target for obesity and related metabolic disturbances. All the findings concerning the regulation of HOXA5 in AT dysfunction from the aforementioned studies are summarized in Supplementary Table S1.
Obesity-related inflammation is widely acknowledged as a contributing factor in various cancers [75]. The presence of HOXA5, which possesses anti-inflammatory properties, plays a crucial role in regulating this inflammatory response. In obese individuals with insufficient HOXA5 levels, this protective anti-inflammatory effect might be compromised. As a result, the chronic inflammatory environment that already exists in obesity could become even more pronounced, leading to a higher risk of cancer development and progression. Indeed, as in obesity, Pay P et al. reported that the loss of HOXA5 increases the pro-inflammatory signaling pathway NF-κB to exacerbate breast cancer aggressiveness [76]. Therefore, HOXA5 deficit may also affect multiple signaling pathways, commonly implicated in connecting obesity with an increased risk of cancer. Consistently, it was reported that the ectopic expression of HOXA5 suppressed proliferation and neoplasia in cervical cancer cells by repressing WNT signaling [77]. In hypertrophic obesity, the silencing of HOXA5 results in an improper activation of this pathway [13,78]. These findings indicate that deregulated HOXA5 expression may serve as a molecular link between metabolic disorders and cancer development. Although this fascinating correlation holds great significance, it remains inadequately explored and warrants further attention.

Changes in HOXA5 DNA Methylation Are Related to Metabolic Diseases
Epigenetic changes have been confirmed to play a critical role in the occurrence and progression of diverse metabolic diseases, including obesity and T2D. A deeper characterization of epigenetic regulatory mechanisms in metabolic diseases allows us to better understand these conditions and develop novel treatment strategies. Epigenetic regulation can occur at various levels, including DNA methylation, histone modifications, and noncoding RNA (ncRNA) modulation. They can all change the expression of genes without affecting their DNA sequence [75]. Histone modifications encompass intricate post-translational alterations like methylation (mono-, di-, and tri-methylation), acetylation, and SUMOylation. Post-translational changes in histone proteins can modify chromatin conformation and regulation of genes involved in glucose and lipid metabolism, thereby contributing to the phenotype of metabolic diseases [79]. NcRNAs have a significant impact on epigenetics, either by altering chromatin structure or controlling gene expression both at the transcriptional and post-transcriptional levels. A wide range of ncRNA types have been discovered, including microRNAs, long ncRNAs, circular RNAs, and novel small ncRNAs derived from known RNAs [80]. These ncRNAs are crucial for maintaining metabolic homeostasis. Dysregulated ncRNA expression is linked to the development and advancement of metabolic disorders [81]. The effects of histone modifications and ncRNA on the development of metabolic diseases have been extensively discussed elsewhere [82] and will not be covered in this review, which is focused on the role of DNA methylation in regulating the HOXA5 gene in the context of metabolic disorders. DNA methylation is a crucial epigenetic modification involving the covalent addition of methyl groups to DNA bases. The process is governed by three conserved enzymes: DNA methyltransferase 1 (DNMT1), DNMT3A, and DNMT3B, which play a pivotal role in its occurrence and maintenance, contributing to normal development. Depending on its location, DNA methylation can lead to gene repression or activation. Typically, when it occurs near the transcription start site or enhancer regions, it suppresses gene expression [17]. The establishment and preservation of the DNA methylation pattern are influenced by various cellular environmental factors, including metabolism (Supplementary Figure S2). One critical pathway involved in this process is the methionine cycle, which converts methionine into S-adenosyl-methionine (SAM), a methyl donor for DNA. After donating the methyl group, SAM is converted back to homocysteine, which can follow two paths: remethylation and transsulfuration [83]. Proper regulation of these pathways is essential to support cellular health and fundamental biological processes. Investigating the transsulfuration pathway could serve as a promising approach to understanding changes in DNA methylation and predicting metabolic diseases, as clearly described by Werge MP et al. [83].
The transcription factor HOXA5 is tightly epigenetically controlled throughout development [84], primarily by DNA methylation. Indeed, abnormal HOXA5 promoter methylation is the most common way by which HOXA5 is dysregulated in developmental diseases and several cancers [5]. In line with the current evidence that DNA methylation alterations in genes regulating AT function may be one of the crucial mechanisms linking obesity to its clinical complications, such as IR and T2D [50,85,86], the HOXA5 gene has been shown to undergo dynamic changes in DNA methylation in metabolic diseases related to AT dysfunction. For example, in mice, a 5-month treatment with an HFD intake caused hyper-methylation of the Hoxa5 promoter, which leads to its downregulation in abdominal fat. Interestingly, replacing HFD with a standard chow diet improves the metabolic phenotype while also restoring normal DNA methylation and expression levels at Hoxa5, suggesting that the Hoxa5 methylation pattern may reflect the obesity response to nutri- tional intervention [11]. Likewise, HOXA5 downregulation observed in pre-adipocytes of FDR individuals was caused by increased DNA methylation at its promoter region, which is significantly associated with signs of dysfunctional SAT, including adipocyte hypertrophy, that characterized these individuals [13]. This supports the concept that aberrant methylation at the promoter of this transcription factor could trigger a completely distinct transcriptional program in the hit tissue, namely, AT, resulting in a wide range of altered downstream transcriptional processes. A study by Rönn T et al., investigating the relationship between DNA methylation and triglyceride levels, found that in human SAT, the methylation of the HOXA5 promoter was significantly associated with levels of circulating triglycerides, implying that these epigenetic changes may mediate the effects of triglycerides on IR [16] (Figure 1). been shown to undergo dynamic changes in DNA methylation in metabolic diseases related to AT dysfunction. For example, in mice, a 5-month treatment with an HFD intake caused hyper-methylation of the Hoxa5 promoter, which leads to its downregulation in abdominal fat. Interestingly, replacing HFD with a standard chow diet improves the metabolic phenotype while also restoring normal DNA methylation and expression levels at Hoxa5, suggesting that the Hoxa5 methylation pattern may reflect the obesity response to nutritional intervention [11]. Likewise, HOXA5 downregulation observed in preadipocytes of FDR individuals was caused by increased DNA methylation at its promoter region, which is significantly associated with signs of dysfunctional SAT, including adipocyte hypertrophy, that characterized these individuals [13]. This supports the concept that aberrant methylation at the promoter of this transcription factor could trigger a completely distinct transcriptional program in the hit tissue, namely, AT, resulting in a wide range of altered downstream transcriptional processes. A study by Rönn T et al., investigating the relationship between DNA methylation and triglyceride levels, found that in human SAT, the methylation of the HOXA5 promoter was significantly associated with levels of circulating triglycerides, implying that these epigenetic changes may mediate the effects of triglycerides on IR [16] (Figure 1). Besides its pathogenetic significance, the DNA methylation level of HOXA5 may also serve as a potential biomarker to identify subjects at increased risk of metabolic disorders, in agreement with the current evidence that methylation changes detected in easily accessible tissues, like blood cells, may serve as a surrogate marker for major metabolic organs directly implicated in disease pathogenesis [87,88]. Indeed, as in pre-adipocytes, we found that the HOXA5 promoter region is hyper-methylated in peripheral blood leukocytes of subjects with T2D familiarity. Importantly, this increased methylation was positively correlated with FDR adipose cell size in SAT, which has been demonstrated to predict T2D [13,89]. These findings indicate that HOXA5 DNA methylation in the blood cells may represent a bona fide surrogate mark of a pre-adipocyte epigenetic profile in FDR. Our study further demonstrated the significance of the HOXA5 methylation profile in blood as a mark of adipose tissue dysfunction and T2D risk since blood methylation at Environmental factors, such as an unbalanced diet and a lack of exercise, can disrupt the correct makeup of DNA methylation at the HOXA5 locus, whose deficit is a distinct feature of a dysfunctional abdominal SAT and may contribute to the development of adverse metabolic outcomes. Figure  created with Biorender.com.
Besides its pathogenetic significance, the DNA methylation level of HOXA5 may also serve as a potential biomarker to identify subjects at increased risk of metabolic disorders, in agreement with the current evidence that methylation changes detected in easily accessible tissues, like blood cells, may serve as a surrogate marker for major metabolic organs directly implicated in disease pathogenesis [87,88]. Indeed, as in pre-adipocytes, we found that the HOXA5 promoter region is hyper-methylated in peripheral blood leukocytes of subjects with T2D familiarity. Importantly, this increased methylation was positively correlated with FDR adipose cell size in SAT, which has been demonstrated to predict T2D [13,89]. These findings indicate that HOXA5 DNA methylation in the blood cells may represent a bona fide surrogate mark of a pre-adipocyte epigenetic profile in FDR. Our study further demonstrated the significance of the HOXA5 methylation profile in blood as a mark of adipose tissue dysfunction and T2D risk since blood methylation at the HOXA5 locus positively correlated with BMI in a group of obese individuals, indicating that the HOXA5 epigenetic profile can also measure the degree of obesity [13,50]. Accordingly, a positive association between DNA methylation in HOXA5 and BMI was reported in a Mexican American cohort, a population with a high prevalence of obesity. An EWAS study by Chilunga et al. examined the relationship between DNA methylation levels and IR in blood samples of an African American cohort, a population characterized by a high risk of developing IR and T2D [90]. Interestingly, in this study population, they found a close association between methylation levels at a specific CpG (Cytosine-phosphate-Guanine) site (cg14013695 at TSS of HOXA5) and IR, measured by the HOMA-IR index [91]. DNA methylation at the same CpG site has previously been associated with T2D-related traits in an EWAS of blood samples from Mexican Americans [92]. Together, these observations not only clearly demonstrate that DNA methylation at the HOXA5 locus plays a role in the pathogenesis of obesity and its related disturbances, such as IR and T2D, but also suggest that the methylation status of HOXA5 may be useful in clinical practice as an epigenetic biomarker in the prediction and diagnosis of metabolic diseases (Table 1).

HOXA5 as a Potential Therapeutic Target
A further promising aspect of epigenetic changes lies in their dynamic nature and potential reversibility under targeted interventions [82]. This ambitious task might be accomplished by modifiable and lifestyle-sensitive epigenetic factors (e.g., nutritional factors, physical activity) or by using epigenetic modifying agents [17]. The FDA (Food and Drug Administration) has not yet approved any drugs targeting epigenetic pathways for the treatment of metabolic disorders. Nonetheless, several epigenetic drugs approved for other diseases, primarily cancer, may potentially work in metabolic diseases [82,93].
As DNA methylation is the primary mechanism by which HOXA5 become dysregulated, research on drugs targeting methylation at the HOXA5 locus appears promising for treating metabolic diseases. DNA methyltransferase inhibitors (DNMTi), which could modulate the methylation levels of specific genes, represent an attractive opportunity for metabolic disorders treatment. For example, the antiarrhythmic drug procainamide, which also acts as an inhibitor of DNMT1, protects against diabetes by restoring the expression of the homeobox PDX1 (Pancreatic and duodenal homeobox 1) gene via a decrease in its methylation levels in β cells [94]. A similar strategy could be used to reduce the HOXA5 promoter methylation in order to restore its function and restrain AT dysfunction in obesity. Another example comes from the decitabine, commonly known as 5-Aza-2 -deoxycytidine, which is a medication approved for the treatment of myelodysplastic syndromes [95]. Decitabine increases the expression of PPARα (Peroxisome proliferator activated receptor alpha) mRNA by regulating DNA methylation levels and reducing lipid accumulation, thereby alleviating non-alcoholic fatty liver disease [96].
Even more interestingly, this compound is also able to improve glucose homeostasis in T2D mice, in part by promoting macrophage polarization to the M2 anti-inflammatory phenotype via increasing PPARγ expression [97], suggesting that decitabine may also be applicable for diabetes therapy. Given the critical role of HOXA5 in promoting the M2 macrophage switch in chronic inflammation, targeting its immunomodulatory action may provide therapeutic benefits and alleviate obesity-related inflammation and metabolic abnormalities. Furthermore, decitabine promotes osteogenic differentiation in a murine model of osteoporosis by decreasing the methylation levels of osteogenic genes, such as RUNX2 (Runt related transcription factor 2). Notable, the expression of RUNX2 is enhanced by WNT2B, which acts as an anti-adipogenic factor and is negatively regulated by HOXA5 [13,98]. Although further research is needed, mainly regarding the adverse effects of these agents on off-target genes, epigenetic modifications specifically targeting HOXA5 appear to be a promising strategy for precision medicine in metabolic diseases.

Conclusions
In this review, we highlighted the relevant role of the transcription factor HOXA5 in the pathogenesis of metabolic diseases. HOXA5 indeed has many faces. It is an essential regulator of adipocyte differentiation and promotes healthy AT expansion and distribution. HOXA5 also ameliorates obesity-related inflammation and metabolic disturbance through its immunomodulatory function. Furthermore, changes in DNA methylation alterations at the HOXA5 gene contribute to adipose cell dysfunction in obese and T2 diabetic individuals. Some of these epigenetic changes are also reflected in blood samples from people who are at high risk of or have metabolic disorders. This renders HOXA5 a potential biomarker applicable to disease prediction and diagnosis. Given the susceptibility of epigenetic modifications to reversal through tailored interventions, targeting this transcription factor for therapeutic purposes might provide novel and realistic opportunities to prevent and treat metabolic diseases.
While this review presents compelling evidence showing that HOXA5 deficiency is a molecular signature of AT dysfunction, a primary factor contributing to metabolic disorders, the exact causal relationship between HOXA5 downregulation and the development of metabolic diseases is yet to be established. To achieve this, it is crucial to understand the specific target genes regulated by HOXA5 that drive its metabolic effects. Additionally, creating an animal model with a targeted deletion of HOXA5 specifically in AT holds promise as an exciting approach to further investigate its role in metabolic disease development.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cells12162090/s1, Figure S1: Flowchart of the systematic review process; Figure S2: Metabolic pathways contributing to DNA methylation; Table S1: Regulation of HOXA5 in different cell types and adipose tissue depots under physiological and metabolically unhealthy conditions, in both humans and murine studies.