Faculty Bibliography

Mibefradil is a tetralol derivative originally developed as an antagonist of T-type voltage-gated calcium (Ca²⁺) channels to treat hypertension when used at nanomolar dosage. More recently, its therapeutic application in hypertension has declined and has been instead repurposed as a treatment of cancer cell proliferation and solid tumor growth. Beyond its function as a Ca_v blocker, the micromolar concentration of mibefradil can stimulate a rise in [Ca²⁺]_cyt although the mechanism is poorly known. The chanzyme TRPM7 (transient receptor potential melastanin 7), the release of intracellular Ca²⁺ pools, and Ca²⁺ influx by ORAI channels have been associated with the increase in [Ca²⁺]_cyt triggered by mibefradil. This study aims to investigate the cellular targets and pathways associated with mibefradil's effect on [Ca²⁺]_cyt. To address these questions, we monitored changes in [Ca²⁺]_cyt in the specialized mouse epithelial cells (LS8 and ALC) and the widely used HEK-293 cells by stimulating these cells with mibefradil (0.1 μM to 100 μM). We show that mibefradil elicits an increase in [Ca²⁺]_cyt at concentrations above 10 μM (IC₅₀ around 50 μM) and a fast Ca²⁺ increase capacity at 100 μM. We found that inhibiting IP₃ receptors, depleting the ER-Ca²⁺ stores, or blocking phospholipase C (PLC), significantly decreased the capacity of mibefradil to elevate [Ca²⁺]_cyt. Moreover, the transient application of 100 μM mibefradil triggered Ca²⁺ influx by store-operated Ca²⁺ entry (SOCE) mediated by the ORAI channels. Our findings reveal that IP₃R and PLC are potential new targets of mibefradil offering novel insights into the effects of this drug.

Most cells use calcium (Ca²⁺) as a second messenger to convey signals that affect a multitude of biological processes. The ability of Ca²⁺ to bind to proteins to alter their charge and conformation is essential to achieve its signaling role. Cytosolic Ca²⁺ (_cCa²⁺) concentration is maintained low at ~100 nM so that the impact of elevations in _cCa²⁺ is readily sensed and transduced by cells. However, such elevations in _cCa²⁺ must be transient to prevent detrimental effects. Cells have developed a variety of systems to rapidly clear the excess of _cCa²⁺ including Ca²⁺ pumps, exchangers and sequestering Ca²⁺ within intracellular organelles. This Ca²⁺ signaling toolkit is evolutionarily adapted so that each cell, tissue, and organ can fulfill its biological function optimally. One of the most specialized cells in mammals are the enamel forming cells, the ameloblasts, which also handle large quantities of Ca²⁺. The end goal of ameloblasts is to synthesize, secrete and mineralize a unique proteinaceous matrix without the benefit of remodeling or repair mechanisms. Ca²⁺ uptake into ameloblasts is mainly regulated by the store operated Ca²⁺ entry (SOCE) before it is transported across the polarized ameloblasts to reach the insulated enamel space. Here we review the ameloblasts Ca²⁺ signaling toolkit and address how the common electronegative non-metal fluoride can alter its function, potentially addressing the biology of dental fluorosis.

Recent studies have provided great insight into hominin life history evolution by utilizing incremental lines found in dental tissues to reconstruct and compare the growth records of extant and extinct humans versus other ape taxa. Among the hominins, studies that have examined Retzius periodicity (RP) variation have come to contradictory conclusions in some instances. To clarify RP variation among hominins and better place this variation in its broader evolutionary context, we conduct the most comprehensive analysis of published RP values for hominins and great apes to date. We gathered all available data from the literature on RP data from extant humans, great apes, and fossil hominins and assessed their variation using parametric and nonparametric analyses of variance. We also performed phylogenetic generalized least-squares regressions of RP data for these taxa as well as a larger set of hominoids for which RP data have been published against data for body mass, encephalization, and mean semicircular canal radius (a proxy for metabolic rate). Our results show that modern humans have a mean RP significantly differing from that of other hominins. Pongo also is significantly different from nearly all other taxa in all analyses. Our results also demonstrate that RP variation among hominins scales with respect to body mass, encephalization, and semicircular canal radius similarly to other hominids but that modern humans and Pongo stand out in this regard. Operating within the hypothesis that RP reflects autonomic biorhythms that regulate multiple life history variables, our results reinforce the idea that Homo sapiens has evolved a life history distinct from other hominins, even from other members of Homo, and suggest that many of these life history differences may be driven by hypothalamic output from the brain.

Characterizing dental development in fossil hominins is important for distinguishing between them and for establishing where and when the slow overall growth and development of modern humans appeared. Dental development of australopiths and early Homo was faster than modern humans. The Atapuerca fossils (Spain) fill a barely known gap in human evolution, spanning ~1.2 to ~0.4 million years (Ma), during which H. sapiens and Neandertal dental growth characteristics may have developed. We report here perikymata counts, perikymata distributions and periodicities of all teeth belonging to the TE9 level of Sima del Elefante, level TD6.2 of Gran Dolina (H. antecessor) and Sima de los Huesos. We found some components of dental growth in the Atapuerca fossils resembled more recent H. sapiens. Mosaic evolution of perikymata counts and distribution generate three distinct clusters: H. antecessor, Sima de los Huesos and H. sapiens.

Calcium (Ca²⁺) release-activated Ca²⁺ (CRAC) channels mediated by STIM1/2 and ORAI (ORAI1-3) proteins form the dominant store-operated Ca²⁺ entry (SOCE) pathway in a wide variety of cells. Among these, the enamel-forming cells known as ameloblasts rely on CRAC channel function to enable Ca²⁺ influx, which is important for enamel mineralization. This key role of the CRAC channel is supported by human mutations and animal models lacking STIM1 and ORAI1, which results in enamel defects and hypomineralization. A number of recent reports have highlighted the role of the chanzyme TRPM7 (transient receptor potential melastanin 7), a transmembrane protein containing an ion channel permeable to divalent cations (Mg²⁺, Ca²⁺), as a modulator of SOCE. This raises the question as to whether TRPM7 should be considered an alternative route for Ca²⁺ influx, or if TRPM7 modifies CRAC channel activity in enamel cells. To address these questions, we monitored Ca²⁺ influx mediated by SOCE using the pharmacological TRPM7 activator naltriben and the inhibitor NS8593 in rat primary enamel cells and in the murine ameloblast cell line LS8 cells stimulated with thapsigargin. We also measured Ca²⁺ dynamics in ORAI1/2-deficient (shOrai1/2) LS8 cells and in cells with siRNA knock-down of Trpm7. We found that primary enamel cells stimulated with the TRPM7 activator potentiated Ca²⁺ influx via SOCE compared to control cells. However, blockade of TRPM7 with NS8593 did not decrease the SOCE peak. Furthermore, activation of TRPM7 in shOrai1/2 LS8 cells lacking SOCE failed to elicit Ca²⁺ influx, and Trpm7 knock-down had no effect on SOCE. Taken together, our data suggest that TRPM7 is a positive modulator of SOCE potentiating Ca²⁺ influx in enamel cells, but its function is fully dependent on the prior activation of the ORAI channels.

Fluoride ions are highly reactive, and their incorporation in forming dental enamel at low concentrations promotes mineralization. In contrast, excessive fluoride intake causes dental fluorosis, visually recognizable enamel defects that can increase the risk of caries. To investigate the molecular bases of dental fluorosis, we analyzed the effects of fluoride exposure in enamel cells to assess its impact on Ca²⁺ signaling. Primary enamel cells and an enamel cell line (LS8) exposed to fluoride showed decreased internal Ca²⁺ stores and store-operated Ca²⁺ entry (SOCE). RNA-sequencing analysis revealed changes in gene expression suggestive of endoplasmic reticulum (ER) stress in fluoride-treated LS8 cells. Fluoride exposure did not alter Ca²⁺ homeostasis or increase the expression of ER stress-associated genes in HEK-293 cells. In enamel cells, fluoride exposure affected the functioning of the ER-localized Ca²⁺ channel IP₃R and the activity of the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump during Ca²⁺ refilling of the ER. Fluoride negatively affected mitochondrial respiration, elicited mitochondrial membrane depolarization, and disrupted mitochondrial morphology. Together, these data provide a potential mechanism underlying dental fluorosis.

Enamel is the most calcified tissue in vertebrates. Enamel formation and mineralization is a two-step process that is mediated by ameloblast cells during their secretory and maturation stages. In these two stages, ameloblasts are characterized by different morphology and function, which is fundamental for proper mineral growth in the extracellular space. Ultrastructural studies have shown that the mitochondria in these cells localize to different subcellular regions in both stages. However, limited knowledge is available on the role/s of mitochondria in enamel formation. To address this issue, we analyzed mitochondrial biogenesis and respiration, as well as the redox status of rat primary enamel cells isolated from the secretory and maturation stages. We show that maturation stage cells have an increased expression of PGC1α, a marker of mitochondrial biogenesis, and of components of the electron transport chain. Oxygen consumption rate (OCR), a proxy for mitochondrial function, showed a significant increase in oxidative phosphorylation during the maturation stage, promoting ATP production. The GSH/GSSG ratio was lower in the maturation stage, indicative of increased oxidation. Because higher oxidative phosphorylation can lead to higher ROS production, we tested if ROS affected the expression of AmelX and Enam genes that are essential for enamel formation. The ameloblast cell line LS8 treated with H₂O₂ to promote ROS elicited significant expression changes in AmelX and Enam. Our data highlight important metabolic and physiological differences across the two enamel stages, with higher ATP levels in the maturation stage indicative of a higher energy demand. Besides these metabolic shifts, it is likely that the enhanced ETC function results in ROS-mediated transcriptional changes.

Calcium signaling plays a central role in bone development and homeostasis. Store operated calcium entry (SOCE) is an important calcium influx pathway mediated by calcium release activated calcium (CRAC) channels in the plasma membrane. Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum calcium sensing protein important for SOCE. We generated a mouse model expressing the STIM1 R304W mutation, causing Stormorken syndrome in humans. Stim1^R304W/R304W mice showed perinatal lethality, and the only three animals that survived into adulthood presented with reduced growth, low body weight, and thoracic kyphosis. Radiographs revealed a reduced number of ribs in the Stim1^R304W/R304W mice. Microcomputed tomography data revealed decreased cortical bone thickness and increased trabecular bone volume fraction in Stim1^R304W/R304W mice, which had thinner and more compact bone compared to wild type mice. The Stim1^R304W/+ mice showed an intermediate phenotype. Histological analyses showed that the Stim1^R304W/R304W mice had abnormal bone architecture, with markedly increased number of trabeculae and reduced bone marrow cavity. Homozygous mice showed STIM1 positive osteocytes and osteoblasts. These findings highlight the critical role of the gain-of-function (GoF) STIM1 R304W protein in skeletal development and homeostasis in mice. Furthermore, the novel feature of bilateral subgingival hair growth on the lower incisors in the Stim1^R304W/R304W mice and 25 % of the heterozygous mice indicate that the GoF STIM1 R304W protein also induces an abnormal epithelial cell fate.

The face is the most distinctive feature used to identify others. Modern humans have a short, retracted face beneath a large globular braincase that is distinctively different from that of our closest living relatives. The face is a skeletal complex formed by 14 individual bones that houses parts of the digestive, respiratory, visual and olfactory systems. A key to understanding the origin and evolution of the human face is analysis of the faces of extinct taxa in the hominin clade over the last 6 million years. Yet, as new fossils are recovered and the number of hominin species grows, the question of how and when the modern human face originated remains unclear. By examining key features of the facial skeleton, here we evaluate the evolutionary history of the modern human face in the context of its development, morphology and function, and suggest that its appearance is the result of a combination of biomechanical, physiological and social influences.