Effect of genetic polymorphisms rs2301113 and rs2057482 in the expression of HIF-1α protein in periodontal ligament fibroblasts subjected to compressive force

Abstract Objective Many genes and signaling molecules are involved in orthodontic tooth movement, with mechanically and hypoxically stabilized HIF-1α having been shown to play a decisive role in periodontal ligament signaling during orthodontic tooth movement. Thus, this in vitro study aimed to investigate if genetic polymorphisms in HIF1A (Hypoxia-inducible factor α-subunits) influence the expression pattern of HIF-1α protein during simulated orthodontic compressive pressure. Methodology Samples from human periodontal ligament fibroblasts were used and their DNA was genotyped using real time Polymerase chain reaction for the genetic polymorphisms rs2301113 and rs2057482 in HIF1A . For cell culture and protein expression experiments, six human periodontal ligament fibroblast cell lines were selected based on the patients’ genotype. To simulate orthodontic compressive pressure in fibroblasts, a 2 g/cm2 force was applied under cell culture conditions for 48 hours. Protein expression was evaluated by Western Blot. Paired t-tests were used to compare HIF-1α expression with and without compressive pressure application and unpaired t-tests were used to compare expression between the genotypes in rs2057482 and rs2301113 (p<0.05). Results The expression of HIF-1α protein was significantly enhanced by compressive pressure application regardless of the genotype (p<0.0001). The genotypes in the genetic polymorphisms rs2301113 and rs2057482 were not associated with HIF-1α protein expression (p>0.05). Conclusions Our study confirms that compressive pressure application enhances HIF-1α protein expression. We could not prove that the genetic polymorphisms in HIF1A affect HIF-1α protein expression by periodontal ligament fibroblasts during simulated orthodontic compressive force.


Introduction
The periodontal ligament (PDL) is a connective tissue attachment located between the cementum of teeth and the alveolar bone. The main PDL cell population comprises fibroblast-like cells characterized by collagen production, but also presenting some osteoblastic features. 1 Fibroblasts are the predominant cells in the PDL and are responsible for the regulation of tissue homoeostasis and the formation of collagenous structural proteins. These cells are also crucial during orthodontic tooth movement (OTM), which is characterized by the application of mechanical force to a tooth by removable or fixed orthodontic appliances. 2 During orthodontic treatment, OTM leads to the formation of tensile and pressure zones in the human periodontal ligament. 3 PDL fibroblasts react to a continuous mechanical pressure with alterations in the expression of many genes important for the regulation and mediation of OTM. [4][5][6][7][8][9] The hypoxia-inducible factor α-subunits (HIFα) are key transcription factors in the mammalian response to oxygen deficiency. 10 HIFα acts as a master regulator of cellular and systemic homeostatic response to hypoxia by activating the transcription of many genes important for OTM, including those involved in angiogenesis and apoptosis. Interestingly, this transcription factor has been highlighted as an important molecule during OTM. 8,11 During OTM, a disruption in PDL vascular circulation occurs at pressure areas of the periodontal ligament due to the compression of blood vessels, leading to hypoxic conditions (reduction in the oxygen supply) in the PDL, stabilizing HIF-1α. 12 Moreover, a recent in vitro study showed that HIF-1α protein levels in PDL fibroblasts were predominantly elevated by simulated orthodontic compressive pressure, suggesting that the main stimulus is mechanical and not hypoxia. 8 The subunit HIF-1α is encoded by the gene HIF1A, which was assigned to the human chromosome 14q21-q24. The HIF1A gene presents some genetic polymorphisms that have been studied in different health conditions and were associated with cancer susceptibility and prognosis, coronary artery disease, and metabolic and cardiovascular risk factors. [13][14][15][16][17][18] Genetic polymorphisms in RANKL (receptor activator of nuclear factor-κB ligand) and COX2 (cyclooxygenase-2) genes associated with OTM were shown to affect the expression of those genes in human PDL (hPDL) fibroblasts during simulated orthodontic compressive pressure. PDL-fibroblast from individuals with specific genotypes in RANKL and COX2 responded differently to in vitro simulated orthodontic compressive pressure. 9 Thus, it is possible to hypothesize that genetic polymorphisms in HIF1A are involved in interindividual differences in HIF-1α levels as a response to OTM.
In this in vitro study, we investigated if the genetic polymorphisms rs2301113 and rs2057482 in HIF1A  Sample collection and selection, DNA isolation, and allelic discrimination analysis PDL samples from 57 patients undergoing dental treatment at the maxillofacial surgery clinic at the University of Regensburg were collected.
Caries-free third molars without periodontal disease extracted during dental treatment were used to collect primary human periodontal ligament (hPDL) fibroblasts from periodontal connective tissue. hPDL samples were collected, isolated, cultivated, and characterized according to an established method and protocol. 5,6 The samples of hPDL fibroblasts for cell culture and protein analysis were selected according to the patients' genotype. Genomic DNA for the allelic discrimination analysis was extracted from these cells.
Briefly, the DNA of hPDL cell samples was isolated using the GenElute Mammalian Genomic DNA Miniprep kit (Sigma Aldrich, Munich, Germany). DNA extraction was performed according to the manufacturer's instructions, as previously described. 19 The two polymorphisms in HIF1A were genotyped with allelic discrimination real-time Polymerase chain reaction Effect of genetic polymorphisms rs2301113 and rs2057482 in the expression of HIF-1α protein in periodontal ligament fibroblasts subjected to compressive force J Appl Oral Sci. 2023;31:e20220151 3/8 (PCR) using the TaqMan assay and the Mastercycler ® ep realplex-S thermocycler (Eppendorf AG, Hamburg, Germany), as previously described. 20 Table 1 shows the characteristics of the genetic polymorphisms.

In vitro-simulated orthodontic compressive pressure
For the cell culture experiment, six hPDL cell lines were selected based on their genotype. To simulate orthodontic compressive pressure in hPDL pressure areas, a 2 g/cm 2 physiological compressive force was applied to the hPDL fibroblasts under cell culture conditions at 70% confluency for 48 h, using a glass disc according to a previously established and published method for simulating orthodontic compressive forces. 5,6,9 At the same time, hPDL fibroblasts were cultivated for 48 h without compressive forces (control).

Protein isolation and Western blot of HIF-1α
The total protein from hPDL was isolated with

Evaluation of the expression of HIF-1α protein
The protein expression analysis showed that HIF-1α was significantly enhanced by simulated orthodontic compressive pressure application regardless of the genotype. Figure 2 shows these results. The graph in Figure   2A shows the comparison of HIF-1α protein expression between no pressure (mean=0.99; Standard deviation=0.04) and during compressive force application (mean=1.64; Standard deviation=0.35).
A statistical significance difference between the groups was observed (p<0.0001). Figure 2B 6,29 It is plausible to assume that the increased levels of HIF-1α observed here are also observed in vivo and followed by an increase in VEGF-A expression.
As previously stated, in vitro studies using hPDL cells are important to identify genes involved in the OTM, but also in the identification of candidate genes for future clinical studies that will aim to identify "slow movers" and "fast movers." 5 In vitro studies with human samples allow us to verify whether genetic polymorphisms may account for mRNA and protein expression and may affect the orthodontic clinical outcome. A previous study showed that a missense However, our study did not support that variations in rs2301113 and rs2057482 play a role in protein expression in OTM and might be not involved in the genetic predisposition for "slow" or "fast" mover phenotypes. However, it is important to emphasize that we investigated genetic polymorphisms located in non-coding region, which do not encode protein sequences, different from the study performed by Kim, et al. 30 (2008).
Our study has some limitations. It is possible that Although a statistical association was not observed in our study, we cannot exclude a more complex genetic basis for the HIF-1α protein expression during OTM. In fact, this transcription factor was significantly increased during in vitro-simulated orthodontic compressive pressure and variation in its expression could be due to genetic polymorphisms and uncommon mutations in HIF1A or in other genes that regulate HIF1A. Further studies are necessary to identify candidate genes for "slow movers" and "fast movers" during orthodontic treatment.