Skeletal mandibular prognathism is characterized by mesial positioning the mandibular first molar relative to the maxillary first molar. Angle Class III malocclusion is associated with numerous combinations of skeletal and dental morphological variations [1]. The prevalence of Angle Class III malocclusion in Caucasians ranges from 0.48% to 4% [2]; however, it exceeds 10% in Japanese individuals [3]. Angle Class III malocclusion can cause speech disorders and impaired mastication. Among adult patients with mandibular prognathism, occasional cases require orthodontic treatment alone, as well as orthognathic surgery [4]. Orthodontic treatment is frequently performed in adult patients with mandibular prognathism. For younger patients with mandibular prognathism, orthopedic treatment using appliances to restrict mandibular growth during the growth phase has been performed. However, in recent years, growth-inhibiting orthodontic treatment has been discouraged because of concerns regarding its impact on temporomandibular joint disorders. Due to genetic factors and other causes, the mandible may grow excessively, leading to the recurrence of mandibular prognathism in the late growth phase. Consequently, some patients may require orthognathic surgery. If orthodontists can predict whether a patient has strong risk factors for excessive mandibular growth, they may be able to select strategies for more effective treatment of mandibular prognathism [5]-[8].

Best vitelliform macular dystrophy (BVMD Best disease) is an autosomal dominant hereditary disorder discovered by Friedrich Best in 1905 and is characterized by the abnormal accumulation of lipofuscin within and beneath retinal pigment epithelial cells. Petrukhin et al. analyzed the best disease families and discovered in 1998 that the cause lies in the retina-specific gene VMD2, located on chromosome 11q13 [9]. VMD2 was renamed Bestrophin-1 (BEST1), and BEST2, BEST3, and BEST4 were identified through homology searches and organized into the Bestrophin family. The Bestrophin family comprises intracellular proteins and proteins detectable in the plasma membrane. They share a common structure, featuring a consensus sequence containing four transmembrane domains, and form pentamers that function on the membrane. Their common function is to act as Ca²⁺-dependent anion channels (e.g., for Cl⁻) [10]-[12].

BEST1 is an integral membrane protein primarily expressed in the retinal pigment epithelium (RPE) and localized in the basal lamina [13]. Within the RPE, BEST1 functions as both an anion channel and a regulator of intracellular calcium signaling [10]. Additionally, BEST1 is expressed in the brain and is involved in the transport of glutamate and GABA [14][15]. On the other hand, BEST2 is known to be involved in the sweating mechanism [16] and the maintenance of intraocular pressure [17].

The BEST3 gene (NM_032735.3) is located on chromosome 12, consisting of 10 exons and 9 introns [18]. The BEST3 protein (NP_116124.2) translated from this gene is approximately 76 kDa and has been confirmed to be expressed in many organs, including skeletal muscle, the brain, the thymus, the adrenal glands, and the stomach [19]. BEST3 differs from other members of the Bestrophin family in that it possesses a long intracellular C-terminus containing a region with a protein kinase G consensus sequence [10] and is ubiquitously distributed throughout various organs. In addition to its previously reported function as a Ca²⁺-dependent anion channel, BEST3 has been found to possess anti-apoptotic functions in arterial smooth muscle cells [20] and anti-inflammatory effects in endothelial cells [21]. Recently, whole-exome analysis of Japanese patients with mandibular prognathism revealed a non-synonymous single nucleotide variant (SNV) in the BEST3 gene [22]. However, the specific function of BEST3 in bone differentiation and metabolism remains unclear. Therefore, in this study, we investigated the effects of the BEST3 gene on the differentiation of undifferentiated mesenchymal cells using cultured mesenchymal cells as a model system.

A whole-exome analysis of Japanese patients with skeletal mandibular prognathism reported the presence of a nonsynonymous single-nucleotide variant of BEST3 [22]. BEST3 mRNA undergoes various alternative splicing processes [18][21][29]-[32]. Among these variants, some lack an ion-conducting domain and their functions remain unknown. Thus, the functional implications of the L606I variant discovered by Kajii et al. [22] remain unclear. Therefore, we conducted this study to test the hypothesis that BEST3 itself may exert some influence on bone formation and metabolism. Human BEST3 cDNA was overexpressed in mouse ST2 cells, because few established human mesenchymal cell lines reproducibly undergo osteoblastic differentiation and mineralization while retaining the characteristics of multipotent undifferentiated mesenchymal cells.

Transient forced expression of pBEST3 leads to ectopic expression of BEST3. This represents approximately one-sixth of the expression level of Actb, which is known to be expressed at relatively high levels, indicating that BEST3 was ectopically expressed at a lower level than Actb (Table 2). One possible concern was that the detected BEST3 amplification reflected amplification of endogenous mouse Best3. This possibility was considered unlikely, because no amplification was observed in the MOCK control group, even after 65 cycles of qPCR. Moreover, the antisense primer shared only 42.9% sequence homology with mouse Best3, making amplification of the endogenous mouse transcript highly improbable (Table 2, lower panel). Overexpression of BEST3 neither promoted calcification in ST2 cells cultured for 3 weeks in α-MEM medium supplemented with β-glycerophosphate and ascorbic acid nor suppressed the promotion of calcification induced by simvastatin (Figure 2). These results suggest several possible explanations. (1) BEST3 drives factors that both induce and suppress calcification accumulation. (2) Although BEST3 induces (or suppresses) the expression of osteoblast differentiation factors, its transient expression prevents maintenance of this phenotype until calcified granules accumulate. (3) BEST3 did not affect ST2 differentiation. To investigate these possibilities, we monitored the gene expression of BMP-2 and BMP-4 [33], proteins that play central roles in bone differentiation, every other day until day 6 of culture—a time point known to maintain sufficient expression even with transient gene transfection (Figure 3A, Figure 3B). The results showed that BEST3 did not cause any significant changes in the expression of Bmp2 or Bmp4. This was also true for the gene expression of osteocalcin [34], a bone-specific non-fibrillar protein (Figure 3C), alkaline phosphatase (Figure 3D), and the pro-α-chain genes of type I and type III collagen (Figure 3E, Figure 3F). These results suggest that the ectopic overexpression of BEST3 does not affect ST2 cell differentiation.

We investigated how BEST3 expression affects the differentiation and activation of osteoclasts, which regulate bone mass changes in vivo [35]. BEST3 overexpression in ST2 cells significantly induced the expression of Tnfsf11, a gene encoding RANKL (Figure 4A, Figure 4B). However, it had little effect on the expression of Tnfrsf11b, which encodes OPG (Figure 4C, Figure 4D). Even after the addition of simvastatin, no significant changes in Tnfsf11 and Tnfrsf11b expression were observed. In contrast, the gene expression of Csf1, the gene encoding M-CSF, showed almost no change with pBEST3 introduction in the absence of simvastatin (Figure 4E), but increased significantly on days 8 - 12 in the presence of simvastatin compared to the absence of simvastatin, and pBEST3 introduction further accentuated this (Figure 4F). A possible explanation for this is that BEST3, like simvastatin, may inhibit the prenylation of Rho to the cell membrane [36]; however, since it did not promote Bmp2 expression (Figure 3A), this possibility appears extremely unlikely. Furthermore, changes in Tnfsf11 and Csf1 expression induced by BEST3 overexpression appeared from day 8 of culture. This suggests the following possibilities: 1: Ectopically overexpressed BEST3 acts on something, which indirectly promotes the expression of Tnfsf11 and Csf1. 2: The decline in transiently overexpressed BEST3 triggers the promotion of Tnfsf11 and Csf1 expression. These mechanisms remain unclear. However, it is clear that BEST3 expression in osteoblasts promotes, at a minimum, the expression of RANKL. Importantly, the increase in Tnfsf11 expression without a corresponding increase in Tnfrsf11b expression is expected to increase the functional RANKL/OPG ratio, thereby favoring osteoclast differentiation and activation. Furthermore, the concomitant increase in Csf1 expression would be expected to further promote osteoclastogenesis by supporting the survival, proliferation, and differentiation of osteoclast precursor cells. Taken together, these findings suggest that, if similar changes occur in vivo, BEST3 overexpression may create a microenvironment favorable for osteoclast formation and bone remodeling. Further research is needed to determine whether this is associated with the genetic factors underlying skeletal mandibular prognathism.

The gene transfer method used in this study was transient transfection rather than stable transfection. Successful BEST3 overexpression was confirmed by RT-qPCR at 48 h after transfection. In proliferating cells, transiently transfected plasmids generally produce maximal mRNA and protein expression within 24 - 72 h after transfection, after which expression gradually declines and is typically lost within approximately 7 days as the plasmid is diluted during cell division [37]. Because we did not directly monitor the duration of BEST3 overexpression throughout the differentiation period, the persistence of transgene expression beyond the early post-transfection phase remains unknown and should be considered a limitation of this study. Nevertheless, the delayed changes in Tnfsf11 and Csf1 expression observed in the present study suggest that transient BEST3 overexpression may initiate downstream regulatory events rather than directly maintaining their expression.

Overexpression of ectopic BEST3 in osteoblast-like ST2 cells promotes RANKL gene and protein expression.

We express our sincere gratitude to the faculty of the Department of Growth and Developmental Dentistry (Division of Orthodontics) for their guidance throughout the study.

This study was approved by the Ohu University Genetic Recombination Safety Committee (2022001-3) and was conducted in accordance with the Ohu University Regulations on the Safety Management of Genetic Recombination Experiments.