The wake- and sleep-modulating neurons of the lateral hypothalamic area demonstrate a differential pattern of degeneration in Alzheimers disease.

BACKGROUND: Sleep-wake dysfunction is an early and common event in Alzheimer's disease (AD). The lateral hypothalamic area (LHA) regulates the sleep and wake cycle through wake-promoting orexinergic neurons (OrxN) and sleep-promoting melanin-concentrating hormone or MCHergic neurons (MCHN). These neurons share close anatomical proximity with functional reciprocity. This study investigated LHA OrxN and MCHN loss patterns in AD individuals. Understanding the degeneration pattern of these neurons will be instrumental in designing potential therapeutics to slow down the disease progression and remediate the sleep-wake dysfunction in AD. METHODS: Postmortem human brain tissue from donors with AD (across progressive stages) and controls were examined using unbiased stereology. Formalin-fixed, celloidin-embedded hypothalamic sections were stained with Orx-A/MCH, p-tau (CP13), and counterstained with gallocyanin. Orx or MCH-positive neurons with or without CP13 inclusions and gallocyanin-stained neurons were considered for stereology counting. Additionally, we extracted RNA from the LHA using conventional techniques. We used customized Neuropathology and Glia nCounter (Nanostring) panels to study gene expression. Wald statistical test was used to compare the groups, and the genes were considered differentially expressed when the p-value was <.05. RESULTS: We observed a progressive decline in OrxN alongside a relative preservation of MCHN. OrxN decreased by 58% (p=0.03) by Braak stages (BB) 1-2 and further declined to 81% (p=0.03) by BB 5-6. Conversely, MCHN demonstrated a non-statistical significant decline (27%, p=0.1088) by BB 6. We observed a progressive increase in differentially expressed genes (DEGs), starting with glial profile changes in BB2. While OrxN loss was observed, Orx-related genes showed upregulation in BB 3-4 compared to BB 0-1. GO and KEGG terms related to neuroinflammatory pathways were mainly enriched. CONCLUSIONS: To date, OrxN loss in the LHA represents the first neuronal population to die preceding the loss of LC neurons. Conversely, MCHN shows resilience to AD p-tau accumulation across Braak stages. The initial loss of OrxN correlates with specific neuroinflammation, glial profile changes, and an overexpression of HCRT, possibly due to hyperexcitation following compensation mechanisms. Interventions preventing OrxN loss and inhibiting p-tau accumulation in the LHA could prevent neuronal loss in AD and, perhaps, the progression of the disease.

Small vs. Large Library Docking for Positive Allosteric Modulators of the Calcium Sensing Receptor.

Drugs acting as positive allosteric modulators (PAMs) to enhance the activation of the calcium sensing receptor (CaSR) and to suppress parathyroid hormone (PTH) secretion can treat hyperparathyroidism but suffer from side effects including hypocalcemia and arrhythmias. Seeking new CaSR modulators, we docked libraries of 2.7 million and 1.2 billion molecules against transforming pockets in the active-state receptor dimer structure. Consistent with simulations suggesting that docking improves with library size, billion-molecule docking found new PAMs with a hit rate that was 2.7-fold higher than the million-molecule library and with hits up to 37-fold more potent. Structure-based optimization of ligands from both campaigns led to nanomolar leads, one of which was advanced to animal testing. This PAM displays 100-fold the potency of the standard of care, cinacalcet, in ex vivo organ assays, and reduces serum PTH levels in mice by up to 80% without the hypocalcemia typical of CaSR drugs. Cryo-EM structures with the new PAMs show that they induce residue rearrangements in the binding pockets and promote CaSR dimer conformations that are closer to the G-protein coupled state compared to established drugs. These findings highlight the promise of large library docking for therapeutic leads, especially when combined with experimental structure determination and mechanism.

Digital spatial profiling of human parathyroid tumors reveals cellular and molecular alterations linked to vitamin D deficiency.

Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder characterized by disrupted calcium homeostasis secondary to inappropriately elevated parathyroid hormone (PTH) secretion. Low levels of serum 25-hydroxyvitamin D (25OHD) are significantly more prevalent in PHPT patients than in the general population (1-3), but the basis for this association remains unclear. We employed a spatially defined in situ whole-transcriptomics and selective proteomics profiling approach to compare gene expression patterns and cellular composition in parathyroid adenomas from vitamin D-deficient or vitamin D-replete PHPT patients. A cross-sectional panel of eucalcemic cadaveric donor parathyroid glands was examined in parallel as normal tissue controls. Here, we report that parathyroid tumors from vitamin D-deficient PHPT patients (Def-Ts) are intrinsically different from those of vitamin D-replete patients (Rep-Ts) of similar age and preoperative clinical presentation. The parathyroid oxyphil cell content is markedly higher in Def-Ts (47.8%) relative to Rep-Ts (17.8%) and normal donor glands (7.7%). Vitamin D deficiency is associated with increased expression of electron transport chain and oxidative phosphorylation pathway components. Parathyroid oxyphil cells, while morphologically distinct, are comparable to chief cells at the transcriptional level, and vitamin D deficiency affects the transcriptional profiles of both cell types in a similar manner. These data suggest that oxyphil cells are derived from chief cells and imply that their increased abundance may be induced by low vitamin D status. Gene set enrichment analysis reveals that pathways altered in Def-Ts are distinct from Rep-Ts, suggesting alternative tumor etiologies in these groups. Increased oxyphil content may thus be a morphological indicator of tumor-predisposing cellular stress.

Impaired Mineral Ion Metabolism in a Mouse Model of Targeted Calcium-Sensing Receptor (CaSR) Deletion from Vascular Smooth Muscle Cells.

BACKGROUND: Impaired mineral ion metabolism is a hallmark of CKD-metabolic bone disorder. It can lead to pathologic vascular calcification and is associated with an increased risk of cardiovascular mortality. Loss of calcium-sensing receptor (CaSR) expression in vascular smooth muscle cells exacerbates vascular calcification in vitro. Conversely, vascular calcification can be reduced by calcimimetics, which function as allosteric activators of CaSR. METHODS: To determine the role of the CaSR in vascular calcification, we characterized mice with targeted Casr gene knockout in vascular smooth muscle cells ( SM22α CaSR Δflox/Δflox ). RESULTS: Vascular smooth muscle cells cultured from the knockout (KO) mice calcified more readily than those from control (wild-type) mice in vitro. However, mice did not show ectopic calcifications in vivo but they did display a profound mineral ion imbalance. Specifically, KO mice exhibited hypercalcemia, hypercalciuria, hyperphosphaturia, and osteopenia, with elevated circulating fibroblast growth factor 23 (FGF23), calcitriol (1,25-D3), and parathyroid hormone levels. Renal tubular α-Klotho protein expression was increased in KO mice but vascular α-Klotho protein expression was not. Altered CaSR expression in the kidney or the parathyroid glands could not account for the observed phenotype of the KO mice. CONCLUSIONS: These results suggest that, in addition to CaSR's established role in the parathyroid-kidney-bone axis, expression of CaSR in vascular smooth muscle cells directly contributes to total body mineral ion homeostasis.

Community-Based Screening for Hepatitis B and C Infectivity Using Two Quantitative Antigens to Identify Endemic Townships.

Screening and linkage to care are essential to achieve viral hepatitis elimination before 2030. The accurate identification of endemic areas is important for controlling diseases with geographic aggregation. Viral activity drives prognosis of chronic hepatitis B and hepatitis C virus infection. This screening was conducted in Chiayi County from 2018-2019. All residents aged 30 years or older were invited to participate in quantitative HBsAg (qHBsAg) and HCV Ag screening. Among the 4010 participants (male:female = 1630:2380), the prevalence of qHBsAg and HCV Ag was 9.9% (396/4010) and 4.1% (163/4010), respectively. High-prevalence townships were identified, three for qHBsAg > 15% and two for HCV Ag > 10%. The age-specific prevalence of qHBsAg was distributed in an inverse U-shape with a peak (16.0%, 68/424) for subjects in their 40 s; for HCV, prevalence increased with age. Concentrations of qHBsAg < 200 IU/mL were found in 54% (214/396) of carriers. The rate of oral antiviral treatment for HCV was 75.5% (114/151), with subjects younger than 75 years tending to undergo treatment (85.6% vs. 57.4%, p < 0.001). QHBsAg and HCV Ag core antigens can reflect the concentration of the viral load, which serves as a feasible screening tool. Using quantitative antigen screening for hepatitis B and C in community-based screening, two hyperendemic townships were identified from an endemic county.

Immune Reconstitution Bone Loss Exacerbates Bone Degeneration Due to Natural Aging in a Mouse Model.

BACKGROUND: Immune reconstitution bone loss (IRBL) is a common side-effect of antiretroviral therapy (ART) in people with human immunodeficiency virus (PWH). Immune reconstitution bone loss acts through CD4+ T-cell/immune reconstitution-induced inflammation and is independent of antiviral regimen. Immune reconstitution bone loss may contribute to the high rate of bone fracture in PWH, a cause of significant morbidity and mortality. Although IRBL is transient, it remains unclear whether bone recovers, or whether it is permanently denuded and further compounds bone loss associated with natural aging. METHODS: We used a validated IRBL mouse model involving T-cell reconstitution of immunocompromised mice. Mice underwent cross-sectional bone phenotyping of femur and/or vertebrae between 6 and 20 months of age by microcomputed tomography (µCT) and quantitative bone histomorphometry. CD4+ T cells were purified at 20 months to quantify osteoclastogenic/inflammatory cytokine expression. RESULTS: Although cortical IRBL in young animals recovered with time, trabecular bone loss was permanent and exacerbated skeletal decline associated with natural aging. At 20 months of age, reconstituted CD4+ T cells express enhanced osteoclastogenic cytokines including RANKL, interleukin (IL)-1ß, IL-17A, and tumor necrosis factor-α, consistent with elevated osteoclast numbers. CONCLUSIONS: Immune reconstitution bone loss in the trabecular compartment is permanent and further exacerbates bone loss due to natural aging. If validated in humans, interventions to limit IRBL may be important to prevent fractures in aging PWH.

Spatial bias in cAMP generation determines biological responses to PTH type 1 receptor activation.

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a class B G protein­coupled receptor (GPCR) that regulates mineral ion, vitamin D, and bone homeostasis. Activation of the PTHR by PTH induces both transient cell surface and sustained endosomal cAMP production. To address whether the spatial (location) or temporal (duration) dimension of PTHR-induced cAMP encodes distinct biological outcomes, we engineered a biased PTHR ligand (PTH7d) that elicits cAMP production at the plasma membrane but not at endosomes. PTH7d stabilized a unique active PTHR conformation that mediated sustained cAMP signaling at the plasma membrane due to impaired ß-arrestin coupling to the receptor. Experiments in cells and mice revealed that sustained cAMP production by cell surface PTHR failed to mimic the pharmacological effects of sustained endosomal cAMP production on the abundance of the rate-limiting hydroxylase catalyzing the formation of active vitamin D, as well as increases in circulating active vitamin D and Ca2+ and in bone formation in mice. Thus, similar amounts of cAMP generated by PTHR for similar lengths of time in different cellular locations, plasma membrane and endosomes, mediate distinct physiological responses. These results unveil subcellular signaling location as a means to achieve specificity in PTHR-mediated biological outcomes and raise the prospect of rational drug design based upon spatiotemporal manipulation of GPCR signaling.

Decreased Calcium-Sensing Receptor Expression Controls Calcium Signaling and Cell-To-Cell Adhesion Defects in Aged Skin.

The calcium-sensing receptor (CaSR) drives essential calcium ion (Ca2+) and E-cadherin‒mediated processes in the epidermis, including differentiation, cell-to-cell adhesion, and epidermal barrier homeostasis in cells and in young adult mice. We now report that decreased CaSR expression leads to impaired Ca2+ signal propagation in aged mouse (aged >22 months) epidermis and human (aged >79 years, donor age) keratinocytes. Baseline cytosolic Ca2+ concentrations were higher, and capacitive Ca2+ entry was lower in aged than in young keratinocytes. As in Casr-knockout mice (EpidCaSR-/-), decreased CaSR expression led to decreased E-cadherin and phospholipase C-γ expression and to a compensatory upregulation of STIM1. Pretreatment with the CaSR agonist N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine normalized Ca2+ propagation and E-cadherin organization after experimental wounding. These results suggest that age-related defects in CaSR expression dysregulate normal keratinocyte and epidermal Ca2+ signaling, leading to impaired E-cadherin expression, organization, and function. These findings show an innovative mechanism whereby Ca2+- and E-cadherin‒dependent functions are impaired in aging epidermis and suggest a new therapeutic approach by restoring CaSR function.

PTH hypersecretion triggered by a GABAB1 and Ca2+-sensing receptor heterocomplex in hyperparathyroidism.

Molecular mechanisms mediating tonic secretion of parathyroid hormone (PTH) in response to hypocalcaemia and hyperparathyroidism (HPT) are unclear. Here we demonstrate increased heterocomplex formation between the calcium-sensing receptor (CaSR) and metabotropic γ-aminobutyric acid (GABA) B1 receptor (GABAB1R) in hyperplastic parathyroid glands (PTGs) of patients with primary and secondary HPT. Targeted ablation of GABAB1R or glutamic acid decarboxylase 1 and 2 in PTGs produces hypocalcaemia and hypoparathyroidism, and prevents PTH hypersecretion in PTGs cultured from mouse models of hereditary HPT and dietary calcium-deficiency. Cobinding of the CaSR/GABAB1R complex by baclofen and high extracellular calcium blocks the coupling of heterotrimeric G-proteins to homomeric CaSRs in cultured cells and promotes PTH secretion in cultured mouse PTGs. These results combined with the ability of PTG to synthesize GABA support a critical autocrine action of GABA/GABAB1R in mediating tonic PTH secretion of PTGs and ascribe aberrant activities of CaSR/GABAB1R heteromer to HPT.

Calcium-Sensing Receptors in Chondrocytes and Osteoblasts Are Required for Callus Maturation and Fracture Healing in Mice.

Calcium and its putative receptor (CaSR) control skeletal development by pacing chondrocyte differentiation and mediating osteoblast (OB) function during endochondral bone formation-an essential process recapitulated during fracture repair. Here, we delineated the role of the CaSR in mediating transition of callus chondrocytes into the OB lineage and subsequent bone formation at fracture sites and explored targeting CaSRs pharmacologically to enhance fracture repair. In chondrocytes cultured from soft calluses at a closed, unfixed fracture site, extracellular [Ca2+ ] and the allosteric CaSR agonist (NPS-R568) promoted terminal differentiation of resident cells and the attainment of an osteoblastic phenotype. Knockout (KO) of the Casr gene in chondrocytes lengthened the chondrogenic phase of fracture repair by increasing cell proliferation in soft calluses but retarded subsequent osteogenic activity in hard calluses. Tracing growth plate (GP) and callus chondrocytes that express Rosa26-tdTomato showed reduced chondrocyte transition into OBs (by >80%) in the spongiosa of the metaphysis and in hard calluses. In addition, KO of the Casr gene specifically in mature OBs suppressed osteogenic activity and mineralizing function in bony calluses. Importantly, in experiments using PTH (1-34) to enhance fracture healing, co-injection of NPS-R568 not only normalized the hypercalcemic side effects of intermittent PTH (1-34) treatment in mice but also produced synergistic osteoanabolic effects in calluses. These data indicate a functional role of CaSR in mediating chondrogenesis and osteogenesis in the fracture callus and the potential of CaSR agonism to facilitate fracture repair. © 2019 American Society for Bone and Mineral Research.

Calcium-sensing receptor deletion in the mouse esophagus alters barrier function.

Calcium-sensing receptor (CaSR) is the molecular sensor by which cells respond to small changes in extracellular Ca2+ concentrations. CaSR has been reported to play a role in glandular and fluid secretion in the gastrointestinal tract and to regulate differentiation and proliferation of skin keratinocytes. CaSR is present in the esophageal epithelium, but its role in this tissue has not been defined. We deleted CaSR in the mouse esophagus by generating keratin 5 CreER;CaSRFlox+/+compound mutants, in which loxP sites flank exon 7 of CaSR gene. Recombination was initiated with multiple tamoxifen injections, and we demonstrated exon 7 deletion by PCR analysis of genomic DNA. Quantitative real-time PCR and Western blot analyses showed a significant reduction in CaSR mRNA and protein expression in the knockout mice (EsoCaSR-/-) as compared with control mice. Microscopic examination of EsoCaSR-/- esophageal tissues showed morphological changes including elongation of the rete pegs, abnormal keratinization and stratification, and bacterial buildup on the luminal epithelial surface. Western analysis revealed a significant reduction in levels of adherens junction proteins E-cadherin and ß catenin and tight junction protein claudin-1, 4, and 5. Levels of small GTPase proteins Rac/Cdc42, involved in actin remodeling, were also reduced. Ussing chamber experiments showed a significantly lower transepithelial resistance in knockout (KO) tissues. In addition, luminal-to-serosal-fluorescein dextran (4 kDa) flux was higher in KO tissues. Our data indicate that CaSR plays a role in regulating keratinization and cell-cell junctional complexes and is therefore important for the maintenance of the barrier function of the esophagus.NEW & NOTEWORTHY The esophageal stratified squamous epithelium maintains its integrity by continuous proliferation and differentiation of the basal cells. Here, we demonstrate that deletion of the calcium-sensing receptor, a G protein-coupled receptor, from the basal cells disrupts the structure and barrier properties of the epithelium.

Calcium-Sensing Receptor Regulates Epidermal Intracellular Ca2+ Signaling and Re-Epithelialization after Wounding.

Extracellular Ca2+ (Ca2+o) is a crucial regulator of epidermal homeostasis and its receptor, the Ca2+-sensing receptor (CaSR), conveys the Ca2+o signals to promote keratinocyte adhesion, differentiation, and survival via activation of intracellular Ca2+ (Ca2+i) and E-cadherin-mediated signaling. Here, we took genetic loss-of-function approaches to delineate the functions of CaSR in wound re-epithelialization. Cutaneous injury triggered a robust CaSR expression and a surge of Ca2+i in epidermis. CaSR and E-cadherin were co-expressed at the cell-cell membrane between migratory keratinocytes in the nascent epithelial tongues. Blocking the expression of CaSR or E-cadherin in cultured keratinocytes markedly inhibited the wound-induced Ca2+i propagation and their ability to migrate collectively. Depleting CaSR also suppressed keratinocyte proliferation by downregulating the E-cadherin/epidermal growth factor receptor/mitogen-activated protein kinase signaling axis. Blunted epidermal Ca2+i response to wounding and retarded wound healing were observed in the keratinocyte-specific CaSR knockout (EpidCasr-/-) mice, whose shortened neo-epithelia exhibited declined E-cadherin expression and diminished keratinocyte proliferation and differentiation. Conversely, stimulating endogenous CaSR with calcimimetic NPS-R568 accelerated wound re-epithelialization through enhancing the epidermal Ca2+i signals and E-cadherin membrane expression. These findings demonstrated a critical role for the CaSR in epidermal regeneration and its therapeutic potential for improving skin wound repair.

Combined Deletion of the Vitamin D Receptor and Calcium-Sensing Receptor Delays Wound Re-epithelialization.

When the skin is injured, keratinocytes proliferate, migrate, and differentiate to regenerate the epidermis. We recently showed that ablation of the vitamin D receptor (Vdr) in keratinocytes delays wound re-epithelialization in mice also fed a low-calcium diet, implicating a cooperative role of Vdr and calcium signaling in this process. In this study, we examined the role of vitamin D and calcium signaling in wound healing by deleting their receptors, Vdr and the calcium-sensing receptor (Casr). Gene expression profiling of neonatal epidermis lacking both Vdr and Casr [Vdr and Casr double knockout (DKO)] specifically in keratinocytes revealed that DKO affects a number of pathways relevant to wound healing, including Vdr, ß-catenin, and adherens junction (AJ) signaling. In adult skin, DKO caused a significant delay in wound closure and re-epithelialization, whereas myofibroblast numbers and matrix deposition were unaffected. The injury-induced proliferation of epidermal keratinocytes was blunted in both epidermis and hair follicles, and expression of ß-catenin target genes was reduced in the DKO. Expression of E-cadherin and desmoglein 1 was reduced in the shortened leading edges of the epithelial tongues re-epithelializing the wounds, consistent with the decreased migration rate of DKO keratinocytes in vitro. These results demonstrate that Vdr and Casr are required for ß-catenin-regulated cell proliferation and AJ formation essential for re-epithelialization after wounding. We conclude that vitamin D and calcium signaling in keratinocytes are required for a normal regenerative response of the skin to wounding.

Disruption of Vitamin D and Calcium Signaling in Keratinocytes Predisposes to Skin Cancer.

1,25 dihydroxyvitamin D (1,25(OH)2D), the active metabolite of vitamin D, and calcium regulate epidermal differentiation. 1,25(OH)2D exerts its effects through the vitamin D receptor (VDR), a transcription factor in the nuclear hormone receptor family, whereas calcium acts through the calcium sensing receptor (Casr), a membrane bound member of the G protein coupled receptor family. We have developed mouse models in which the Vdr and Casr have been deleted in the epidermis ((epid) Vdr (-∕-) and (epid) Casr (-∕-)). Both genotypes show abnormalities in calcium induced epidermal differentiation in vivo and in vitro, associated with altered hedgehog (HH) and ß-catenin signaling that when abnormally expressed lead to basal cell carcinomas (BCC) and trichofolliculomas, respectively. The Vdr (-∕-) mice are susceptible to tumor formation following UVB or chemical carcinogen exposure. More recently we found that the keratinocytes from these mice over express long non-coding RNA (lncRNA) oncogenes such as H19 and under express lncRNA tumor suppressors such as lincRNA-21. Spontaneous tumors have not been observed in either the (epid) Vdr (-∕-) or (epid) Casr (-∕-). But in mice with epidermal specific deletion of both Vdr and Casr ((epid) Vdr (-∕-)/(epid) Casr (-∕-) [DKO]) tumor formation occurs spontaneously when the DKO mice are placed on a low calcium diet. These results demonstrate important interactions between vitamin D and calcium signaling through their respective receptors that lead to cancer when these signals are disrupted. The roles of the ß-catenin, hedgehog, and lncRNA pathways in predisposing the epidermis to tumor formation when vitamin D and calcium signaling are disrupted will be discussed.

Interplay between CaSR and PTH1R signaling in skeletal development and osteoanabolism.

Parathyroid hormone (PTH)-related peptide (PTHrP) controls the pace of pre- and post-natal growth plate development by activating the PTH1R in chondrocytes, while PTH maintains mineral and skeletal homeostasis by modulating calciotropic activities in kidneys, gut, and bone. The extracellular calcium-sensing receptor (CaSR) is a member of family C, G protein-coupled receptor, which regulates mineral and skeletal homeostasis by controlling PTH secretion in parathyroid glands and Ca(2+) excretion in kidneys. Recent studies showed the expression of CaSR in chondrocytes, osteoblasts, and osteoclasts and confirmed its non-redundant roles in modulating the recruitment, proliferation, survival, and differentiation of the cells. This review emphasizes the actions of CaSR and PTH1R signaling responses in cartilage and bone and discusses how these two signaling cascades interact to control growth plate development and maintain skeletal metabolism in physiological and pathological conditions. Lastly, novel therapeutic regimens that exploit interrelationship between the CaSR and PTH1R are proposed to produce more robust osteoanabolism.

Vitamin D and calcium regulation of epidermal wound healing.

Wound healing is essential for survival. This is a multistep process involving a number of different cell types. In the skin wounding triggers an acute inflammatory response, with the innate immune system contributing both to protection against invasive organisms and to triggering the invasion of inflammatory cells into the wounded area. These cells release a variety of cytokines and growth factors that stimulate the proliferation and migration of dermal and epidermal cells to close the wound. In particular, wounding activates stem cells in the interfollicular epidermis (IFE) and hair follicles (HF) to proliferate and send their progeny to re-epithelialize the wound. ß-catenin and calcium signaling are important for this activation process. Mice lacking the VDR when placed on a low calcium diet have delayed wound healing. This is associated with reduced ß-catenin transcriptional activity and proliferation in the cells at the leading edge of wound closure. These data suggest that vitamin D and calcium signaling are necessary components of the epidermal response to wounding, likely by regulating stem cell activation through increased ß-catenin transcriptional activity.

Novel mechanisms for the vitamin D receptor (VDR) in the skin and in skin cancer.

The VDR acting with or without its principal ligand 1,25(OH)2D regulates two central processes in the skin, interfollicular epidermal (IFE) differentiation and hair follicle cycling (HFC). Calcium is an important co-regulator with 1,25(OH)2D at least of epidermal differentiation. Knockout of the calcium sensing receptor (CaSR) in addition to VDR accelerates the development of skin cancer in mice on a low calcium diet. Coactivators such as mediator 1 (aka DRIP205) and steroid receptor coactivator 3 (SRC3) regulate VDR function at different stages of the differentiation process, with Med 1 essential for hair follicle differentiation and early stages of epidermal differentiation and proliferation and SRC3 essential for the latter stages of differentiation including formation of the permeability barrier and innate immunity. The corepressor of VDR, hairless (HR), is essential for hair follicle cycling, although its effect on epidermal differentiation in vivo is minimal. In its regulation of HFC and IFE VDR controls two pathways-wnt/ß-catenin and sonic hedgehog (SHH). In the absence of VDR these pathways are overexpressed leading to tumor formation. Whereas, VDR binding to ß-catenin may block its activation of TCF/LEF1 sites, ß-catenin binding to VDR may enhance its activation of VDREs. 1,25(OH)2D promotes but may not be required for these interactions. Suppression of SHH expression by VDR, on the other hand, requires 1,25(OH)2D. The major point of emphasis is that the role of VDR in the skin involves a number of novel mechanisms, both 1,25(OH)2D dependent and independent, that when disrupted interfere with IFE differentiation and HFC, predisposing to cancer formation. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Calcium-sensing receptor (CaSR) as a novel target for ischemic neuroprotection.

OBJECT: Ischemic brain injury is the leading cause for death and long-term disability in patients who suffer cardiac arrest and embolic stroke. Excitotoxicity and subsequent Ca(2+)-overload lead to ischemic neuron death. We explore a novel mechanism concerning the role of the excitatory extracellular calcium-sensing receptor (CaSR) in the induction of ischemic brain injury. METHOD: Mice were exposed to forebrain ischemia and the actions of CaSR were determined after its genes were ablated specifically in hippocampal neurons or its activities were inhibited pharmacologically. Since the CaSR forms a heteromeric complex with the inhibitory type B γ-aminobutyric acid receptor 1 (GABABR1), we compared neuronal responses to ischemia in mice deficient in CaSR, GABABR1, or both, and in mice injected locally or systemically with a specific CaSR antagonist (or calcilytic) in the presence or absence of a GABABR1 agonist (baclofen). RESULTS: Both global and focal brain ischemia led to CaSR overexpression and GABABR1 downregulation in injured neurons. Genetic ablation of Casr genes or blocking CaSR activities by calcilytics rendered robust neuroprotection and preserved learning and memory functions in ischemic mice, partly by restoring GABABR1 expression. Concurrent ablation of Gabbr1 gene blocked the neuroprotection caused by the Casr gene knockout. Coinjection of calcilytics with baclofen synergistically enhanced neuroprotection. This combined therapy remained robust when given 6 h after ischemia. INTERPRETATION: Our study demonstrates a novel receptor interaction, which contributes to ischemic neuron death through CaSR upregulation and GABABR1 downregulation, and feasibility of neuroprotection by concurrently targeting these two receptors.

Obligatory roles of filamin A in E-cadherin-mediated cell-cell adhesion in epidermal keratinocytes.

BACKGROUND: Extracellular Ca(2+) (Cao(2+))-induced E-cadherin-mediated cell-cell adhesion plays a critical role in promoting differentiation in epidermal keratinocytes. Our previous studies show that the calcium-sensing receptor (CaR) regulates keratinocyte cell-cell adhesion and differentiation via Rho A-mediated signaling. CaR forms a protein complex with Rho A, guanine nucleotide exchange factor Trio, and a cytoskeletal actin-binding protein, filamin A, at the cell-cell junctions in response to elevated Cao(2+) levels. Filamin A has the ability to interact directly with CaR, Trio, and Rho and mediate CaR-dependent signaling events. OBJECTIVE: This study was conducted to investigate the roles of filamin A and Trio in regulating Cao(2+)-induced Rho activation and intercellular adhesion. METHODS: Expression of filamin A and Trio in keratinocytes was inhibited by siRNA. Its effects on Cao(2+)-dependent junction formation and adhesion complex formation were evaluated by fluorescence immunostaining and immunoprecipitation. Endogenous Rho activity and expression of keratinocyte differentiation markers were also examined. The significance of the physical interactions of filamin A with Trio and Rho was assessed in dominant-negative inhibition studies. RESULTS: Inhibiting filamin A expression blocked the formation of CaR-Rho A-Trio-E-cadherin protein complex. Knockdown of filamin A or Trio inhibited Cao(2+)-induced membrane localization and activation of Rho A, formation of the E-cadherin-catenin adhesion complex, and keratinocyte terminal differentiation. Expressing dominant-negative peptides disruptive to the endogenous filamin-Trio, filamin-Rho, and CaR-filamin interactions suppressed the formation of adherens junctions. CONCLUSION: Through physical interactions with CaR, Trio and Rho, filamin A generates a scaffold for organizing a signaling complex that promotes E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.

Role of the calcium-sensing receptor in calcium regulation of epidermal differentiation and function.

The epidermis is a stratified squamous epithelium composed of proliferating basal and differentiated suprabasal keratinocytes. It serves as the body's major physical and chemical barrier against infection and harsh environmental insults, as well as preventing excess water loss from the body into the atmosphere. Calcium is a key regulator of the proliferation and differentiation in keratinocytes. Elevated extracellular Ca(2+) concentration ([Ca(2+)]o) raises the levels of intracellular free calcium ([Ca(2+)]i), promotes cell-cell adhesion, and activates differentiation-related genes. Keratinocytes deficient in the calcium-sensing receptor fail to respond to [Ca(2+)]o stimulation and to differentiate, indicating a role for the calcium-sensing receptor in transducing the [Ca(2+)]o signal during differentiation. The concepts derived from in vitro gene knockdown experiments have been evaluated and confirmed in three mouse models in vivo.