Rodrigo S. Lacruz

Lamellar Bone is an Incremental Tissue Reconciling Enamel Rhythms, Body Size, and Organismal Life History

Mammalian enamel formation is periodic, including fluctuations attributable to the daily biological clock as well as longer-period oscillations that enigmatically correlate with body mass. Because the scaling of bone mass to body mass is an axiom of vertebrate hard tissue biology, we consider that long-period enamel formation rhythms may reflect corresponding and heretofore unrecognized rhythms in bone growth. The principal aim of this study is to seek a rhythm in bone growth demonstrably related to enamel oscillatory development. Our analytical approach is based in morphology, using a variety of hard tissue microscopy techniques. We first ascertain the relationship among long-period enamel rhythms, the striae of Retzius, and body mass using a large sample of mammalian taxa. In addition, we test whether osteocyte lacuna density (a surrogate for rates of cell proliferation) in bone is correlated with mammalian body mass. Finally, using fluorescently labeled developing bone tissues, we investigate whether the bone lamella, a fundamental microanatomical unit of bone, relates to rhythmic enamel growth increments. Our results confirm a positive correlation between long-period enamel rhythms and body mass and a negative correlation between osteocyte density and body mass. We also confirm that lamellar bone is an incremental tissue, one lamella formed in the species-specific time dependency of striae of Retzius formation. We conclude by contextualizing our morphological research with a current understanding of autonomic regulatory control of the skeleton and body mass, suggesting a central contribution to the coordination of organismal life history and body mass.

Diseases caused by mutations in ORAI1 and STIM1

Ca2+ release‐activated Ca2+ (CRAC) channels mediate a specific form of Ca2+ influx called store‐operated Ca2+ entry (SOCE) that contributes to the function of many cell types. CRAC channels are composed of ORAI1 proteins located in the plasma membrane, which form its ion‐conducting pore. ORAI1 channels are activated by stromal interaction molecule (STIM) 1 and STIM2 located in the endoplasmic reticulum. Loss‐ and gain‐of‐function gene mutations in ORAI1 and STIM1 in human patients cause distinct disease syndromes. CRAC channelopathy is caused by loss‐of‐function mutations in ORAI1 and STIM1 that abolish CRAC channel function and SOCE; it is characterized by severe combined immunodeficiency (SCID)‐like disease, autoimmunity, muscular hypotonia, and ectodermal dysplasia, with defects in sweat gland function and dental enamel formation. The latter defect emphasizes an important role of CRAC channels in tooth development. By contrast, autosomal dominant gain‐of‐function mutations in ORAI1 and STIM1 result in constitutive CRAC channel activation, SOCE, and increased intracellular Ca2+ levels that are associated with an overlapping spectrum of diseases, including nonsyndromic tubular aggregate myopathy (TAM) and York platelet and Stormorken syndromes. The latter two syndromes are defined, besides myopathy, by thrombocytopenia, thrombopathy, and bleeding diathesis. The fact that myopathy results from both loss‐ and gain‐of‐function mutations in ORAI1 and STIM1 highlights the importance of CRAC channels for Ca2+ homeostasis in skeletal muscle function. The cellular dysfunction and clinical disease spectrum observed in mutant patients provide important information about the molecular regulation of ORAI1 and STIM1 proteins and the role of CRAC channels in human physiology.

Regulation of pH During Amelogenesis

During amelogenesis, extracellular matrix proteins interact with growing hydroxyapatite crystals to create one of the most architecturally complex biological tissues. The process of enamel formation is a unique biomineralizing system characterized first by an increase in crystallite length during the secretory phase of amelogenesis, followed by a vast increase in crystallite width and thickness in the later maturation phase when organic complexes are enzymatically removed. Crystal growth is modulated by changes in the pH of the enamel microenvironment that is critical for proper enamel biomineralization. Whereas the genetic bases for most abnormal enamel phenotypes (amelogenesis imperfecta) are generally associated with mutations to enamel matrix specific genes, mutations to genes involved in pH regulation may result in severely affected enamel structure, highlighting the importance of pH regulation for normal enamel development. This review summarizes the intra- and extracellular mechanisms employed by the enamel-forming cells, ameloblasts, to maintain pH homeostasis and, also, discusses the enamel phenotypes associated with disruptions to genes involved in pH regulation.

The Sodium Bicarbonate Cotransporter (NBCe1) Is Essential for Normal Development of Mouse Dentition

Proximal renal tubular acidosis (pRTA) is a syndrome caused by abnormal proximal tubule reabsorption of bicarbonate resulting in metabolic acidosis. Patients with mutations to the SLC4A4 gene (coding for the sodium bicarbonate cotransporter NBCe1), have pRTA, growth delay, ocular defects, and enamel abnormalities. In an earlier report, we provided the first evidence that enamel cells, the ameloblasts, express NBCe1 in a polarized fashion, thereby contributing to trans-cellular bicarbonate transport. To determine whether NBCe1 plays a critical role in enamel development, we studied the expression of NBCe1 at various stages of enamel formation in wild-type mice and characterized the biophysical properties of enamel in NBCe1−/− animals. The enamel of NBCe1−/− animals was extremely hypomineralized and weak with an abnormal prismatic architecture. The expression profile of amelogenin, a known enamel-specific gene, was not altered in NBCe1−/− animals. Our results show for the first time that NBCe1 expression is required for the development of normal enamel. This study provides a mechanistic model to account for enamel abnormalities in certain patients with pRTA.

Requirements for Ion and Solute Transport, and pH Regulation During Enamel Maturation

Transcellular bicarbonate transport is suspected to be an important pathway used by ameloblasts to regulate extracellular pH and support crystal growth during enamel maturation. Proteins that play a role in amelogenesis include members of the ABC transporters (SLC gene family and CFTR). A number of carbonic anhydrases (CAs) have also been identified. The defined functions of these genes are likely interlinked during enamel mineralization. The purpose of this study is to quantify relative mRNA levels of individual SLC, Cftr, and CAs in enamel cells obtained from secretory and maturation stages on rat incisors. We also present novel data on the enamel phenotypes for two animal models, a mutant porcine (CFTR‐ΔF508) and the NBCe1‐null mouse. Our data show that two SLCs (AE2 and NBCe1), Cftr, and Car2, Car3, Car6, and Car12 are all significantly up‐regulated at the onset of the maturation stage of amelogenesis when compared to the secretory stage. The remaining SLCs and CA gene transcripts showed negligible expression or no significant change in expression from secretory to maturation stages. The enamel of CFTR‐ΔF508 adult pigs was hypomineralized and showed abnormal crystal growth. NBCe1‐null mice enamel was structurally defective and had a marked decrease in mineral content relative to wild‐type. These data demonstrate the importance of many non‐matrix proteins to amelogenesis and that the expression levels of multiple genes regulating extracellular pH are modulated during enamel maturation in response to an increased need for pH buffering during hydroxyapatite crystal growth. J. Cell. Physiol. 227: 1776–1785, 2012.

Identification of novel candidate genes involved in mineralization of dental enamel by genome-wide transcript profiling.

The gene repertoire regulating vertebrate biomineralization is poorly understood. Dental enamel, the most highly mineralized tissue in mammals, differs from other calcifying systems in that the formative cells (ameloblasts) lack remodeling activity and largely degrade and resorb the initial extracellular matrix. Enamel mineralization requires that ameloblasts undergo a profound functional switch from matrix‐secreting to maturational (calcium transport, protein resorption) roles as mineralization progresses. During the maturation stage, extracellular pH decreases markedly, placing high demands on ameloblasts to regulate acidic environments present around the growing hydroxyapatite crystals. To identify the genetic events driving enamel mineralization, we conducted genome‐wide transcript profiling of the developing enamel organ from rat incisors and highlight over 300 genes differentially expressed during maturation. Using multiple bioinformatics analyses, we identified groups of maturation‐associated genes whose functions are linked to key mineralization processes including pH regulation, calcium handling, and matrix turnover. Subsequent qPCR and Western blot analyses revealed that a number of solute carrier (SLC) gene family members were up‐regulated during maturation, including the novel protein Slc24a4 involved in calcium handling as well as other proteins of similar function (Stim1). By providing the first global overview of the cellular machinery required for enamel maturation, this study provide a strong foundation for improving basic understanding of biomineralization and its practical applications in healthcare. J. Cell. Physiol. 227: 2264–2275, 2012.

New Paradigms on the Transport Functions of Maturation-stage Ameloblasts

Fully matured dental enamel is an architecturally and mechanically complex hydroxyapatite-based bioceramic devoid of most of the organic material that was essential in its making. Enamel formation is a staged process principally involving secretory and maturation stages, each associated with major changes in gene expression and cellular function. Cellular activities that define the maturation stage of amelogenesis include ion (e.g., calcium and phosphate) transport and storage, control of intracellular and extracellular pH (e.g., bicarbonate and hydrogen ion movements), and endocytosis. Recent studies on rodent amelogenesis have identified a multitude of gene products that appear to be linked to these cellular activities. This review describes the main cellular activities of these genes during the maturation stage of amelogenesis.

The circadian clock modulates enamel development.

Fully mature enamel is about 98% mineral by weight. While mineral crystals appear very early during its formative phase, the newly secreted enamel is a soft gel-like matrix containing several enamel matrix proteins of which the most abundant is amelogenin (Amelx). Histological analysis of mineralized dental enamel reveals markings called cross-striations associated with daily increments of enamel formation, as evidenced by injections of labeling dyes at known time intervals. The daily incremental growth of enamel has led to the hypothesis that the circadian clock might be involved in the regulation of enamel development. To identify daily rhythms of clock genes and Amelx, we subjected murine ameloblast cells to serum synchronization to analyze the expression of the circadian transcription factors Per2 and Bmal1 by real-time PCR. Results indicate that these key genetic regulators of the circadian clock are expressed in synchronized murine ameloblast cell cultures and that their expression profile follows a circadian pattern with acrophase and bathyphase for both gene transcripts in antiphase. Immunohistological analysis confirms the protein expression of Bmal and Cry in enamel cells. Amelx expression in 2-day postnatal mouse molars dissected every 4 hours for a duration of 48 hours oscillated with an approximately 24-hour period, with a significant approximately 2-fold decrease in expression during the dark period compared to the light period. The expression of genes involved in bicarbonate production (Car2) and transport (Slc4a4), as well as in enamel matrix endocytosis (Lamp1), was greater during the dark period, indicating that ameloblasts express these proteins when Amelx expression is at the nadir. The human and mouse Amelx genes each contain a single nonconserved E-box element within 10 kb upstream of their respective transcription start sites. We also found that within 2 kb of the transcription start site of the human NFYA gene, which encodes a positive regulator of amelogenin, there is an E-box element that is conserved in rodents and other mammals. Moreover, we found that Nfya expression in serum-synchronized murine ameloblasts oscillated with a strong 24-hour rhythm. Taken together, our data support the hypothesis that the circadian clock temporally regulates enamel development.

Primate enamel evinces long period biological timing and regulation of life history

The factor(s) regulating the combination of traits that define the overall life history matrix of mammalian species, comprising attributes such as brain and body weight, age at sexual maturity, lifespan and others, remains a complete mystery. The principal objectives of the present research are (1) to provide evidence for a key variable effecting life history integration and (2) to provide a model for how one would go about investigating the metabolic mechanisms responsible for this rhythm. We suggest here that a biological rhythm with a period greater than the circadian rhythm is responsible for observed variation in primate life history. Evidence for this rhythm derives from studies of tooth enamel formation. Enamel contains an enigmatic periodicity in its microstructure called the striae of Retzius, which develops at species specific intervals in units of whole days. We refer to this enamel rhythm as the repeat interval (RI). For primates, we identify statistically significant relationships between RI and all common life history traits. Importantly, RI also correlates with basal and specific metabolic rates. With the exception of estrous cyclicity, all relationships share a dependence upon body mass. This dependence on body mass informs us that some aspect of metabolism is responsible for periodic energy allocations at RI timescales, regulating cell proliferation rates and growth, thus controlling the pace, patterning, and co-variation of life history traits. Estrous cyclicity relates to the long period rhythm in a body mass-independent manner. The mass-dependency and -independency of life history relationships with RI periodicity align with hypothalamic-mediated neurosecretory anterior and posterior pituitary outputs. We term this period the Havers-Halberg Oscillation (HHO), in reference to Clopton Havers, a 17th Century hard tissue anatomist, and Franz Halberg, a long-time explorer of long-period rhythms. We propose a mathematical model that may help elucidate the underlying physiological mechanism responsible for the HHO.

Adaptor protein complex 2–mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis

Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast‐mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real‐time PCR, we show that the expression of clathrin and adaptor protein subunits are upregulated in maturation stage rodent enamel organ cells. AP complex 2 (AP‐2) is the most upregulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP‐2 and clathrin in ameloblasts, with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP‐2 mediated, clathrin‐dependent endocytosis, thus implying the likelihood of specific membrane‐bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis‐related gene products is also upregulated during maturation including: lysosomal‐associated membrane protein 1 (Lamp1); cluster of differentiation 63 and 68 (Cd63 and Cd68); ATPase, H+ transporting, lysosomal V0 subunit D2 (Atp6v0d2); ATPase, H+ transporting, lysosomal V1 subunit B2 (Atp6v1b2); chloride channel, voltage‐sensitive 7 (Clcn7); and cathepsin K (Ctsk). Immunohistologic data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63‐null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63‐null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast‐like LS8 cells treated with the enamel matrix protein complex Emdogain showed upregulation of AP‐2 and clathrin subunits, further supporting the existence of a membrane‐bound receptor‐regulated pathway for the endocytosis of enamel matrix proteins. These data together define an endocytotic pathway likely used by ameloblasts to remove the enamel matrix during enamel maturation.