Faculty Bibliography

Store-operated Ca²⁺ entry (SOCE) channels are highly selective Ca²⁺ channels activated by the endoplasmic reticulum (ER) sensors STIM1 and STIM2. Their direct interaction with the pore-forming plasma membrane ORAI proteins (ORAI1, ORAI2, and ORAI3) leads to sustained Ca²⁺ fluxes that are critical for many cellular functions. Mutations in the human ORAI1 gene result in immunodeficiency, anhidrotic ectodermal dysplasia, and enamel defects. In our investigation of the role of ORAI proteins in enamel, we identified enamel defects in a patient with an ORAI1 null mutation. Targeted deletion of the Orai1 gene in mice showed enamel defects and reduced SOCE in isolated enamel cells. However, Orai2^-/- mice showed normal enamel despite having increased SOCE in the enamel cells. Knockdown experiments in the enamel cell line LS8 suggested that ORAI2 and ORAI3 modulated ORAI1 function, with ORAI1 and ORAI2 being the main contributors to SOCE. ORAI1-deficient LS8 cells showed altered mitochondrial respiration with increased oxygen consumption rate and ATP, which was associated with altered redox status and enhanced ER Ca²⁺ uptake, likely due to S-glutathionylation of SERCA pumps. Our findings demonstrate an important role of ORAI1 in Ca²⁺ influx in enamel cells and establish a link between SOCE, mitochondrial function, and redox homeostasis.

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca²⁺ influx via Ca²⁺ release-activated Ca²⁺ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca²⁺ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca²⁺ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.

Biomineralization requires the controlled movement of ions across cell barriers to reach the sites of crystal growth. Mineral precipitation occurs in aqueous phases as fluids become supersaturated with specific ionic compositions. In the biological world, biomineralization is dominated by the presence of calcium (Ca²⁺) in crystal lattices. Ca²⁺ channels are intrinsic modulators of this process, facilitating the availability of Ca²⁺ within cells in a tightly regulated manner in time and space. Unequivocally, the most mineralized tissue produced by vertebrates, past and present, is dental enamel. With some of the longest carbonated hydroxyapatite (Hap) crystals known, dental enamel formation is fully coordinated by specialized epithelial cells of ectodermal origin known as ameloblasts. These cells form enamel in two main developmental stages: a) secretory; and b) maturation. The secretory stage is marked by volumetric growth of the tissue with limited mineralization, and the opposite is found in the maturation stage, as enamel crystals expand in width concomitant with increased ion transport. Disruptions in the formation and/or mineralization stages result, in most cases, in permanent alterations in the crystal assembly. This introduces weaknesses in the material properties affecting enamel's hardness and durability, thus limiting its efficacy as a biting, chewing tool and increasing the possibility of pathology. Here, we briefly review enamel development and discuss key properties of ameloblasts and their Ca²⁺-handling machinery, and how alterations in this toolkit result in enamelopathies.

Enamel mineralization relies on Ca²⁺ availability provided by Ca²⁺ release activated Ca²⁺ (CRAC) channels. CRAC channels are modulated by the endoplasmic reticulum Ca²⁺ sensor STIM1 which gates the pore subunit of the channel known as ORAI1, found the in plasma membrane, to enable sustained Ca²⁺ influx. Mutations in the STIM1 and ORAI1 genes result in CRAC channelopathy, an ensemble of diseases including immunodeficiency, muscular hypotonia, ectodermal dysplasia with defects in sweat gland function and abnormal enamel mineralization similar to amelogenesis imperfecta (AI). In some reports, the chief medical complain has been the patient's dental health, highlighting the direct and important link between CRAC channels and enamel. The reported enamel defects are apparent in both the deciduous and in permanent teeth and often require extensive dental treatment to provide the patient with a functional dentition. Among the dental phenotypes observed in the patients, discoloration, increased wear, hypoplasias (thinning of enamel) and chipping has been reported. These findings are not universal in all patients. Here we review the mutations in STIM1 and ORAI1 causing AI-like phenotype, and evaluate the enamel defects in CRAC channel deficient mice. We also provide a brief overview of the role of CRAC channels in other mineralizing systems such as dentine and bone.

Cutaneous T cell lymphoma is a heterogeneous group of lymphomas characterized by the accumulation of malignant T cells in the skin. The molecular and cellular etiology of this malignancy remains enigmatic and what role antigenic stimulation plays in the initiation and/or progression of the disease remains to be elucidated. Deep sequencing of the tumor genome revealed a highly heterogeneous landscape of genetic perturbations and transcriptome analysis of transformed T cells further highlighted the heterogeneity of this disease. Nonetheless, using data harvested from high-throughput transcriptional profiling allowed us to develop a reliable signature of this malignancy. Focusing on a key cytokine signaling pathway, previously implicated in CTCL pathogenesis, JAK/STAT signaling, we used conditional gene targeting to develop a fully penetrant small animal model of this disease that recapitulates many key features of mycosis fungoides, a common variant of CTCL. Using this mouse model, we demonstrate that T cell receptor engagement is critical for malignant transformation of the T lymphocytes and that progression of the disease is dependent on microbiota.

Modern humans have smaller faces relative to Middle and Late Pleistocene members of the genus Homo. While facial reduction and differences in shape have been shown to increase biting efficiency in Homo sapiens relative to these hominins, facial size reduction has also been said to decrease our ability to resist masticatory loads. This study compares crania of Homo heidelbergensis and H. sapiens with respect to mechanical advantages of masticatory muscles, force production efficiency, strains experienced by the cranium and modes of deformation during simulated biting. Analyses utilize X-ray computed tomography (CT) scan-based 3D models of a recent modern human and two H. heidelbergensis. While having muscles of similar cross-sectional area to H. heidelbergensis, our results confirm that the modern human masticatory system is more efficient at converting muscle forces into bite forces. Thus, it can produce higher bite forces than Broken Hill for equal muscle input forces. This difference is the result of alterations in relative in and out-lever arm lengths associated with well-known differences in midfacial prognathism. Apparently at odds with this increased efficiency is the finding that the modern human cranium deforms more, resulting in greater strain magnitudes than Broken Hill when biting at the equivalent tooth. Hence, the facial reduction that characterizes modern humans may not have evolved as a result of selection for force production efficiency. These findings provide further evidence for a degree of uncoupling between form and function in the masticatory system of modern humans. This may reflect the impact of food preparation technologies. These data also support previous suggestions that differences in bite force production efficiency can be considered a spandrel, primarily driven by the midfacial reduction in H. sapiens that occurred for other reasons. Midfacial reduction plausibly resulted in a number of other significant changes in morphology, such as the development of a chin, which has itself been the subject of debate as to whether or not it represents a mechanical adaptation or a spandrel.

Dental enamel is formed by specialized epithelial cells which handle large quantities of Ca²⁺ while producing the most highly mineralized tissue. However, the mechanisms used by enamel cells to handle bulk Ca²⁺ safely remain unclear. Our previous work contradicted the dogma that Ca²⁺ is ferried through the cytosol of Ca²⁺-transporting cells and instead suggested an organelle-based route across enamel cells. This new paradigm involves endoplasmic reticulum (ER)-associated Ca²⁺ stores and their concomitant refilling by store-operated Ca²⁺ entry (SOCE) mediated by Ca²⁺ release activated Ca²⁺ (CRAC) channels. Given that Ca²⁺ handling is maximal during the enamel-mineralization stage (maturation), we anticipated that SOCE would also be elevated then. Confirmation was obtained here using single-cell recordings of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in rat ameloblasts. A candidate SOCE agonist, cholecystokinin (CCK), was found to be upregulated during maturation, with Cck transcript abundance reaching 30% of that in brain. CCK-receptor transcripts were also detected and Ca²⁺ imaging showed that CCK stimulation increased [Ca²⁺]_cyt in a dose-responsive manner that was sensitive to CRAC-channel inhibitors. Similar effects were observed with two other SOCE activators, acetylcholine and ATP, whose receptors were also found in enamel cells. These results provide the first evidence of a potential regulatory system for SOCE in enamel cells and so strengthen the Ca²⁺ transcytosis paradigm for ER-based transport of bulk Ca²⁺. Our findings also implicate enamel cells as a new physiological target of CCK and raise the possibility of an auto/paracrine system for regulating Ca²⁺ transport.

The Encouraging Novel Amelogenesis Models and Ex vivo cell Lines (ENAMEL) Development workshop was held on 23 June 2017 at the Bethesda headquarters of the National Institute of Dental and Craniofacial Research (NIDCR). Discussion topics included model organisms, stem cells/cell lines, and tissues/3D cell culture/organoids. Scientists from a number of disciplines, representing institutions from across the United States, gathered to discuss advances in our understanding of enamel, as well as future directions for the field.

Dental enamel is the hardest and most mineralized tissue in extinct and extant vertebrate species and provides maximum durability that allows teeth to function as weapons and/or tools as well as for food processing. Enamel development and mineralization is an intricate process tightly regulated by cells of the enamel organ called ameloblasts. These heavily polarized cells form a monolayer around the developing enamel tissue and move as a single forming front in specified directions as they lay down a proteinaceous matrix that serves as a template for crystal growth. Ameloblasts maintain intercellular connections creating a semi-permeable barrier that at one end (basal/proximal) receives nutrients and ions from blood vessels, and at the opposite end (secretory/apical/distal) forms extracellular crystals within specified pH conditions. In this unique environment, ameloblasts orchestrate crystal growth via multiple cellular activities including modulating the transport of minerals and ions, pH regulation, proteolysis, and endocytosis. In many vertebrates, the bulk of the enamel tissue volume is first formed and subsequently mineralized by these same cells as they retransform their morphology and function. Cell death by apoptosis and regression are the fates of many ameloblasts following enamel maturation, and what cells remain of the enamel organ are shed during tooth eruption, or are incorporated into the tooth's epithelial attachment to the oral gingiva. In this review, we examine key aspects of dental enamel formation, from its developmental genesis to the ever-increasing wealth of data on the mechanisms mediating ionic transport, as well as the clinical outcomes resulting from abnormal ameloblast function.

Enamel is the most calcified tissue in vertebrates. It differs from bone in a number of characteristics including its origin from ectodermal epithelium, lack of remodeling capacity by the enamel forming cells, and absence of collagen. The enamel-forming cells known as ameloblasts, choreograph first the synthesis of a unique protein-rich matrix, followed by the mineralization of this matrix into a tissue that is approximately 95% mineral. To do this, ameloblasts arrange the coordinated movement of ions across a cell barrier while removing matrix proteins and monitoring extracellular pH using a variety of buffering systems to enable the growth of carbonated apatite crystals. Although our knowledge of these processes and the molecular identity of the proteins involved in transepithelial ion transport has increased in the last decade, it remains limited compared to other cells. Here we present an overview of the evolution and development of enamel, its differences with bone, and describe the ion transport systems associated with ameloblasts.