The research team, led by Wenyan Ruan, Jing Dong, Yali Xu and senior author Xiaohong Duan of the Fourth Military Medical University in Xi’an, China, began with a deceptively simple observation. Salivary glands and teeth share a common ectodermal origin, and both depend on chloride and bicarbonate transport to do their jobs. Saliva secretion is driven primarily by transepithelial chloride transport, which generates the high intracellular chloride concentration that acinar cells need to produce fluid. Enamel maturation, meanwhile, requires precise pH regulation mediated by bicarbonate exchange. SLC4A9, a sodium-dependent chloride/bicarbonate exchanger belonging to the solute carrier family 4, is highly expressed in both the submandibular gland and in amelobasts, the enamel-forming cells. That dual expression raised an intriguing possibility: could dysfunction of this one transporter simultaneously disrupt salivary secretion and enamel mineralization?

The genetic evidence came first. Immunohistochemical analysis of minor salivary gland specimens from five women with primary Sjögren’s syndrome and six control subjects with non-Sjögren’s dry mouth revealed robust SLC4A9 immunoreactivity in the ductal and acinar epithelial compartments of the controls. In the Sjögren’s patients, however, SLC4A9 expression was markedly reduced in both glandular epithelium and infiltrating mononuclear cells, with only residual staining detectable. Whole-genome sequencing of one patient uncovered a heterozygous copy number variant, a deletion within an intronic region of SLC4A9 on chromosome 5. Bioinformatic analysis of ENCODE chromatin data showed that this deleted region contains two constitutively occupied binding sites for CTCF, an insulator protein that organizes three-dimensional chromatin structure. The loss of these CTCF anchoring sites, the authors propose, likely disrupts the native chromatin configuration of SLC4A9 and reduces its expression, suggesting that the transporter’s deficiency may be a primary driver of disease rather than a secondary consequence of inflammation.

To test causality, the team generated Slc4a9 knockout mice using CRISPR/Cas9 targeting that removed exons 3 through 14 of the gene. The results were striking. When salivary flow was stimulated with pilocarpine, the knockout mice secreted approximately 78.6 percent less saliva than wild-type controls in females and about 50 percent less in males. Histology revealed degenerative changes throughout the submandibular gland: ductal epithelial cells showed irregular nuclear contours, aberrant chromatin distribution and partial nuclear pyknosis, while acinar cells displayed vacuolation and atrophy. Transmission electron microscopy exposed a secretory apparatus in collapse, with secretory granules that were fewer in number, enlarged in size and scattered away from their normal apical clustering toward basal and perinuclear regions. Periodic acid-Schiff staining showed excessive mucin retention, and transcriptomic analysis confirmed upregulation of multiple mucin genes, including the gel-forming mucins Muc6, Muc10 and Muc19 and the membrane-associated mucins Muc1, Muc3a and Muc24, alongside dysregulated glycoconjugate metabolism.

With age, the knockout mice developed something even more telling: a Sjögren’s-like autoimmune phenotype. Focal lymphocytic infiltration appeared in the salivary glands of female knockout mice as early as six months of age and expanded by ten months, while males showed a delayed and milder onset. Serological profiling revealed age-progressive autoantibody production. Female knockouts exhibited significantly elevated titres of anti-nuclear antibodies, anti-SSA/Ro and anti-SSB/La from six months onward, mirroring the diagnostic hallmarks of human Sjögren’s syndrome. O-glycomic profiling of salivary proteins further revealed profound remodeling of glycosylation sites on immunoregulatory molecules such as ASM3A, HYOU1 and DYHC1, changes that gene ontology analysis linked to innate and adaptive immune pathways. Because patients with Sjögren’s syndrome also show aberrant glycosylation of salivary mucins like MUC5B and MUC7, which affects saliva viscosity and mucosal clearance, the mouse model captured features of the human disease that extend well beyond simple fluid secretion.

The teeth told the other half of the story. Adult knockout mice displayed chalky, fracture-prone incisors with irregular wear, cuspal delamination and attrition in molars, and disrupted dentin-enamel boundaries. Micro-computed tomography confirmed significantly reduced enamel thickness, and scanning electron microscopy revealed disorganized hydroxyapatite crystallites with porous, loosely packed enamel rods that had lost their normal interwoven architecture. Crucially, the defect was developmental, not merely the consequence of a dry mouth. At postnatal day 1, knockout molars lacked discernible enamel matrix entirely, with ameloblasts arrested in a pre-secretory state and devoid of Tomes’ processes, the specialized extensions that secretory-stage ameloblasts use to deposit enamel. Single-cell RNA sequencing of first molar tooth germs reconstructed the ameloblast differentiation trajectory and showed a blockade: pre-ameloblasts accumulated while secretory-stage ameloblasts dwindled. Enamel matrix genes such as Amelx, Ambn, Enam, Mmp20 and Klk4 were downregulated, whereas dentin markers DSPP and DMP1 were unaffected, indicating a selective failure of the enamel biomineralization program. Primary tooth germ cells from knockouts also formed fewer mineralized nodules under calcification-inducing conditions.

The mechanistic thread connecting these two tissues proved to be ion transport itself. Functional assays using the fluorescent chloride indicator MQAE showed that primary salivary gland cells from knockout mice completely lost the time-dependent chloride uptake response seen in wild-type cells under high-chloride conditions. In tooth germ cells, chloride transport activity was similarly blunted, and measurements with the pH-sensitive dye BCECF revealed that knockout cells could no longer alkalinize their cytosol in response to chloride withdrawal. Transport activity dropped by roughly 45 percent in heterozygous and 55 percent in homozygous knockout cells. Importantly, the related exchanger SLC4A2 showed no compensatory upregulation, underscoring the non-redundant role of SLC4A9 in maintaining the chloride and pH homeostasis that both saliva secretion and enamel mineralization demand.

What happens next inside the cell is where the study makes its most unexpected leap. Prior work had hinted that chloride levels can influence ribosomal genes, and the team’s transcriptomic data showed that RPS27, a component of the 40S ribosomal small subunit, was among the ribosome-related molecules differentially expressed in knockout tissues. In wild-type mice, RPS27 displayed a polarized, punctate apical distribution with minimal nuclear signal. In knockout salivary glands and tooth germs, that organization collapsed: RPS27 accumulated diffusely in the cytoplasm and increased in the nucleus, as confirmed by western blotting of fractionated proteins. The number of B23-positive nucleoli, a readout of ribosome biogenesis, dropped significantly in both tissues, indicating nucleolar stress. Cell-cycle analysis of single-cell sequencing data showed an accumulation of pre-ameloblasts in S phase, a signature of p53-dependent arrest, and the p53-MDM2 axis was visibly perturbed, with p53 and MDM2 increasing in the cytoplasm while decreasing in the nucleus.

The therapeutic payoff came from Nutlin-3a, a selective MDM2 antagonist. The researchers treated knockout mice beginning at four and six months of age, the window before overt Sjögren’s-like pathology, administering 20 milligrams per kilogram every 48 hours for one month. Histological examination at seven months showed that lymphocytic infiltration was dramatically reduced in treated animals compared with saline controls, and the excessive accumulation of PAS-reactive material, which diastase digestion confirmed was largely glycogen, returned to near-control levels. Serum anti-SSA antibody levels also fell, particularly in females. The result validates the RPS27-MDM2-p53 axis as a druggable node and suggests that spatially targeted MDM2 inhibitors could one day complement the immunosuppressive approaches that have so far failed to restore exocrine function in Sjögren’s patients.

Human genetics closed the loop. Whole-exome sequencing of three patients with amelogenesis imperfecta from two Chinese families identified two missense variants in SLC4A9, c.899G>T (p.Arg300Leu) in the two siblings and c.344G>A (p.Arg115Gln) in the sporadic case, both predicted damaging and both affecting arginine residues that are 100 percent conserved from human to rat. Structural modeling showed that these substitutions abolish key hydrogen bonds that stabilize the protein’s cytoplasmic region. When the mutants were expressed in LS8 ameloblast-like cells, chloride uptake fell significantly below control levels, the ribosomal marker RPL7A shifted into the nucleus, and RPS27 upregulation with p53-MDM2 activation recapitulated the mouse findings. Notably, the three patients showed no significant reduction in salivary secretion, while the Sjögren’s patient with the SLC4A9 deletion had normal enamel, indicating that the two phenotypes arise independently through the same gene. The authors propose that SLC4A9 acts as a monogenic cause of amelogenesis imperfecta, likely through dominant-negative effects, while contributing to Sjögren’s syndrome through more complex mechanisms including haploinsufficiency. Together, the work establishes a unified diagnostic target for two seemingly unrelated diseases and points toward interventions, from MDM2 inhibitors to potential chloride supplementation or gene delivery, that could restore both salivary function and enamel integrity.

Subject of Research: The role of SLC4A9 chloride/bicarbonate exchanger deficiency in Sjögren's syndrome and amelogenesis imperfecta via ribosomal stress

Article Title: SLC4A9 deficiency is involved in Sjögren’s syndrome and enamel defects through RPS27-triggered ribosomal stress

Article References: Ruan, W., Dong, J., Xu, Y., Zhang, Y., Zhu, Y., Mao, S., Liu, Y., & Duan, X. (2026). SLC4A9 deficiency is involved in Sjögren’s syndrome and enamel defects through RPS27-triggered ribosomal stress. Experimental & Molecular Medicine. https://doi.org/10.1038/s12276-026-01867-0

Image Credits: AI Generated

DOI: 10.1038/s12276-026-01867-0

Keywords: SLC4A9, Sjögren's syndrome, amelogenesis imperfecta, RPS27, ribosomal stress, p53, MDM2, Nutlin-3a, salivary gland, enamel mineralization, chloride transport, autoantibodies

Salivary basal cell adenoma represents a unique clinical and pathological entity distinguished by its indolent behavior and characteristic morphological features. Despite its benign nature, the tumor’s precise cellular origins and the genetic alterations that trigger its formation have been elusive. Wong and colleagues have focused their lens on the Wnt/β-catenin cascade, an evolutionarily conserved signaling pathway instrumental in regulating cell proliferation, differentiation, and stem cell maintenance. Aberrant activation of this pathway is well-documented in various cancers; however, its role in salivary gland tumors has been relatively unexplored until this seminal study.

The research team employed a combination of next-generation sequencing, immunohistochemical staining, and functional assays on tumor specimens and cell cultures derived from patients diagnosed with BCA. Their data revealed a striking pattern: hotspot mutations in either FBXW11, which encodes an E3 ubiquitin ligase component involved in β-catenin degradation, or CTNNB1, the gene encoding β-catenin itself, are mutually exclusive, meaning that tumors harbor mutations in one gene or the other, but not both. This exclusivity underscores a common oncogenic mechanism converging on dysregulated Wnt/β-catenin signaling.

At the molecular level, FBXW11 mutations impair the formation or function of the β-catenin destruction complex. Under normal circumstances, this complex tags β-catenin for ubiquitin-mediated proteasomal degradation, thus maintaining tight control over intracellular β-catenin levels. In mutant tumors, however, ineffective β-catenin degradation leads to its accumulation in the cytoplasm and nucleus. Concurrently, activating mutations in CTNNB1 stabilize β-catenin by altering critical phosphorylation sites, rendering it resistant to degradation. Both scenarios culminate in enhanced transcriptional activity of β-catenin target genes that promote cellular proliferation and survival—hallmarks of tumorigenesis.

Immunohistochemical analyses provided compelling visual confirmation of these molecular disturbances. Tumors with FBXW11 mutations exhibited robust nuclear localization of β-catenin, contrasting starkly with adjacent normal salivary gland tissues that showed predominantly membranous β-catenin staining. Similarly, CTNNB1 mutant tumors exhibited aberrant nuclear and cytoplasmic β-catenin accumulation. These findings cement the role of improper β-catenin regulation in the pathogenesis of BCA and provide a reliable biomarker profile that can aid pathologists in differential diagnosis.

The functional significance of the discovered mutations was further elucidated through in vitro experiments. When wild-type salivary gland epithelial cells were engineered to express mutant FBXW11 or CTNNB1, they demonstrated enhanced proliferation rates, resistance to apoptosis, and morphological changes consistent with neoplastic transformation. Moreover, RNA sequencing of these engineered cells revealed upregulation of canonical Wnt target genes such as MYC, CCND1 (Cyclin D1), and AXIN2, confirming activation of downstream oncogenic programs.

Beyond characterizing the genetic landscape of BCA, the study tackled the therapeutic implications of these findings. Small molecule inhibitors targeting the Wnt/β-catenin pathway were tested on primary tumor cells harboring either mutation. These agents effectively decreased nuclear β-catenin levels and suppressed cell viability, highlighting a promising therapeutic vulnerability. Given that current management of BCA largely involves surgical excision with limited adjunctive treatments, this pharmacologic insight heralds a paradigm shift towards molecularly tailored therapies.

The mutually exclusive nature of FBXW11 and CTNNB1 mutations raises intriguing questions about tumor heterogeneity and clonal evolution. It suggests a model in which either mutation is sufficient to perturb β-catenin regulation and initiate tumor formation. The study also addresses potential mechanisms of mutation exclusivity, proposing that the presence of one mutation creates a cellular milieu inappropriate for the survival or selection of a second, functionally redundant mutation. This insight adds nuance to our understanding of intratumoral genetic architecture and may influence the design of biomarker panels for precise molecular diagnosis.

One of the striking outcomes of this research is the potential refinement of diagnostic criteria for salivary gland tumors. With overlapping histological features among various salivary gland neoplasms, molecular profiling is increasingly crucial. The identification of FBXW11 and CTNNB1 hotspot mutations as signatures of BCA could establish a genetic hallmark, improving diagnostic accuracy, especially in challenging cases where morphology alone is insufficient. Additionally, nuclear β-catenin immunohistochemistry may serve as a useful adjunct tool in routine pathology practice.

The interplay between tumor genetics and the Wnt/β-catenin signaling cascade in BCA also sheds light on fundamental aspects of salivary gland biology. The pathway is known to orchestrate developmental processes in glandular tissues, and its dysregulation in neoplasia reflects a hijacking of embryonic programs to promote aberrant growth. Understanding these developmental parallels not only contextualizes BCA within a broader biological framework but also raises prospects for leveraging developmental signaling modulators in therapeutic approaches.

Furthermore, the study’s reliance on cutting-edge genomic and proteomic technologies underscores the transformative impact of these tools in cancer biology. Deep sequencing enabled the precise mapping of mutation hotspots, while advanced imaging and cell-based assays verified their functional consequences. The integration of multidisciplinary methodologies exemplifies the modern approach to dissecting complex tumorigenic processes and sets a blueprint for future investigations.

Notably, the significance of FBXW11—also known as β-TrCP2—in cancer biology extends beyond salivary glands. It participates in the ubiquitination and degradation of multiple substrates implicated in cell cycle progression and signal transduction. Its mutation-driven dysfunction may therefore have ripple effects on other oncogenic pathways, hinting at wider relevance that transcends the current tumor model. Similarly, CTNNB1 mutations are well-studied in diverse malignancies, reaffirming the centrality of β-catenin as a master regulator in oncogenesis.

The robust experimental design and comprehensive analysis presented by Wong et al. embody a significant leap forward in our mechanistic understanding of salivary gland tumors. By pinpointing the molecular switch that ignites basal cell adenoma, this research not only demystifies a rare tumor entity but also demonstrates the clinical potential of targeting Wnt/β-catenin signaling. Future directions envisioned include expanding patient cohorts to validate these findings across populations and exploring combination therapies that augment Wnt pathway inhibition.

In sum, the identification of mutually exclusive FBXW11 and CTNNB1 hotspot mutations as key drivers of salivary basal cell adenoma exemplifies the power of molecular medicine to unravel the complexity of tumor biology. This landmark discovery reshapes the diagnostic landscape, informs therapeutic strategies, and enriches our comprehension of tissue-specific oncogenesis. As research continues to illuminate the intricate genetic choreography behind cancer, studies like this propel us closer to precision oncology tailored to the unique molecular signatures of individual tumors.

Subject of Research: Molecular mechanisms driving salivary basal cell adenoma via Wnt/β-catenin pathway activation through FBXW11 and CTNNB1 hotspot mutations.

Article Title: Wnt/β-catenin activation by mutually exclusive FBXW11 and CTNNB1 hotspot mutations drives salivary basal cell adenoma.

Article References:
Wong, K., Bishop, J.A., Weinreb, I. et al. Wnt/β-catenin activation by mutually exclusive FBXW11 and CTNNB1 hotspot mutations drives salivary basal cell adenoma. Nat Commun 16, 4657 (2025). https://doi.org/10.1038/s41467-025-59871-3

Image Credits: AI Generated