Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

Retinoids, vitamin A derivatives, are important regulators of the growth and differentiation of skin cells. Although retinoids are therapeutically used for several skin ailments, little is known about their effects on P2 receptors, known to be involved in various functions in the skin. DNA array analysis showed that treatment of normal human epidermal keratinocytes (NHEKs) with all-trans-retinoic acid (ATRA), an agonist to RAR (retinoic acid receptor), enhanced the expression of mRNA for the P2Y2 receptor, a metabotropic P2 receptor that is known to be involved in the proliferation of the epidermis. The expression of other P2 receptors in NHEKs was not affected by ATRA. ATRA increased the mRNA for the P2Y2 receptor in a concentration-dependent fashion (1 nM to 1 μM). Am80, a synthesized agonist to RAR, showed a similar enhancement, whereas 9-cis-retinoic acid (9-cisRA), an agonist to RXR (retinoid X receptor), enhanced P2Y2 gene expression to a lesser extent. Ca²⁺ imaging analysis showed that ATRA also increased the function of P2Y2 receptors in NHEKs. Retinoids are known to enhance the turnover of the epidermis by increasing both proliferation and terminal differentiation. The DNA microarray analysis also revealed that ATRA upregulates various genes involved in the differentiation of NHEKs. Our present results suggest that retinoids, at least in part, exert their proliferative effects by upregulating P2Y2 receptors in NHEKs. This effect of retinoids may be closely related to their therapeutic effect against various ailments or aging events in skins such as over-keratinization, pigmentation and re-modeling.     

P2 receptors in atherosclerosis and postangioplasty restenosis

Atherosclerosis is an immunoinflammatory process that involves complex interactions between the vessel wall and blood components and is thought to be initiated by endothelial dysfunction [1–3]. Extracellular nucleotides that are released from a variety of arterial and blood cells [4] can bind to P2 receptors and modulate proliferation and migration of smooth muscle cells (SMC), which is known to be involved in intimal hyperplasia that accompanies atherosclerosis and postangioplasty restenosis [5]. In addition, P2 receptors mediate many other functions, including platelet aggregation, leukocyte adherence, and arterial vasomotoricity. A direct pathological role of P2 receptors is reinforced by recent evidence showing that up-regulation and activation of P2Y₂ receptors in rabbit arteries mediates intimal hyperplasia [6]. In addition, up-regulation of functional P2Y receptors also has been demonstrated in the basilar artery of the rat double-hemorrhage model [7] and in coronary arteries of diabetic dyslipidemic pigs [8]. It has been proposed that up-regulation of P2Y receptors may be a potential diagnostic indicator for the early stages of atherosclerosis [9]. Therefore, particular effort must be made to understand the consequences of nucleotide release from cells in the cardiovascular system and the subsequent effects of P2 nucleotide receptor activation in blood vessels, which may reveal novel therapeutic strategies for atherosclerosis and restenosis after angioplasty.     

A genetic screen for behavioral mutations that perturb dopaminergic homeostasis in mice

Disruption of dopaminergic (DA) systems is thought to play a central role in the addictive process and in the pathophysiology of schizophrenia. Although inheritance plays an important role in the predisposition to these disorders, the genetic basis of this is not well understood. To provide additional insight, we have performed a modifier screen in mice designed to identify mutations that perturb DA homeostasis. With a genetic background sensitized by a mutation in the dopamine transporter (DAT), we used random chemical mutagenesis and screened for mutant mice with locomotor abnormalities. Four mutant lines were identified with quantitatively elevated levels of locomotor activity. Mapping of mutations in these lines identified two loci that alter activity only when dopamine levels are elevated by a DAT mutation and thus would only have been uncovered by this type of approach. One of these quantitative trait loci behaves as an enhancer of DA neurotransmission, whereas the other may act as a suppressor. In addition, we also identified three loci which are not dependent on the sensitized background but which also contribute to the overall locomotor phenotype.     

Olfactory receptors and odour coding

The first step of olfactory detection involves interactions between odorant molecules and neuronal protein receptors. Odour coding results from the combinatory activation of a set of receptors and rests on their clonal expression and olfactory neurone connexion, which lead to formation of a specific sensory map in the cortex. This system, sufficient to discriminate myriads of odorants with a mere 350 different receptors, allows humans to smell molecules that are not natural (new cooking flavours, synthetic chemicals...). The extreme olfactory genome diversity explains the absence of odour semantics. Olfactory receptors are also involved in cellular chemotaxis.     

Generation of a mouse for conditional excision of progesterone receptor

The progesterone receptor (PR) is required for several aspects of mammalian female reproduction. PR null mice have overlapping defects that preclude an understanding of its multiple functions in ovulation, pregnancy, mammary gland biology, and sexual behavior. We have generated a PR conditional excision (PRCE) allele in which loxP sites flank exon 1. Homozygous PRCE females are fertile and appear to be functionally normal. Global cre mediated excision of the floxed exon 1 using EIIa-cre mice resulted in systemic loss of exon 1 and PR protein. Female mice homozygous for this null allele were sterile, as expected for PR knockout (PRKO) females. Conditional loss of PR will facilitate investigation of the spatial and temporal roles of PR in both normal development and disease.     

LPA induces osteoblast differentiation through interplay of two receptors: LPA1 and LPA4

The bioactive phospholipid, lysophosphatidic acid (LPA), acting through at least five distinct receptors LPA1–LPA5, plays important roles in numerous biological processes. Here we report that LPA induces osteoblastic differentiation of human mesenchymal stem cells hMSC‐TERT. We find that hMSC‐TERT mostly express two LPA receptors, LPA1 and LPA4, and undergo osteoblastic differentiation in serum‐containing medium. Inhibition of LPA1 with Ki16425 completely abrogates osteogenesis, indicating that this process is mediated by LPA in the serum through activation of LPA1. In contrast to LPA1, down‐regulation of LPA4 expression with shRNA significantly increases osteogenesis, suggesting that this receptor normally exerts negative effects on differentiation. Mechanistically, we find that in hMSC‐TERT, LPA induces a rise in both cAMP and Ca²⁺. The rise in Ca²⁺ is completely abolished by Ki16425, whereas LPA‐mediated cAMP increase is not sensitive to Ki16425. To test if LPA signaling pathways controlling osteogenesis in vitro translate into animal physiology, we evaluated the bones of LPA4‐deficient mice. Consistent with the ability of LPA4 to inhibit osteoblastic differentiation of stem cells, LPA4‐deficient mice have increased trabecular bone volume, number, and thickness. J. Cell. Biochem. 109: 794–800, 2010. © 2010 Wiley‐Liss, Inc.     

House dust mite extract activates apical Cl− channels through protease‐activated receptor 2 in human airway epithelia

Adequate fluid secretion from airway mucosa is essential for maintaining mucociliary clearance, and fluid hypersecretion is a prominent feature of inflammatory airway diseases such as allergic rhinitis. House dust mite extract (HDM) has been reported to activate protease‐activated receptors (PARs), which play various roles in airway epithelia. However, the role of HDM in regulating ion transporters and fluid secretion has not been investigated. We examined the effect of HDM on ion transport in human primary nasal epithelial cells. The Ca²⁺‐sensitive dye Fura2‐AM was used to determine intracellular Ca²⁺ concentration ([Ca²⁺]_i) by means of spectrofluorometry in human normal nasal epithelial cells (NHNE). Short‐circuit current (Isc) was measured using Ussing chambers. Fluid secretion from porcine airway mucosa was observed by optical measurement. HDM extract (10 µg/Ml) effectively cleaved the PAR‐2 peptide and induced an increase of [Ca²⁺]_i that was abolished by desensitization with trypsin, but not with thrombin. Apical application of HDM‐induced Isc sensitive to both a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and a Ca²⁺‐activated Cl^? channel (CaCC) inhibitor. HDM extract also stimulated fluid secretion from porcine airway mucosa. HDM extract activated PAR‐2 and apical Cl^? secretion via CaCC and CFTR, and HDM‐induced fluid secretion in porcine airway mucosa. Our results suggest a role for PAR‐2 in mucociliary clearance and fluid hypersecretion of airway mucosa in response to air‐borne allergens such as HDM. J. Cell. Biochem. 109: 1254–1263, 2010. © 2010 Wiley‐Liss, Inc.     

不同鼠龄的人多巴胺D5^F173L突变基因转基因高血压小鼠血压及心脏结构与功能分析

目的通过对不同年龄多巴胺D5^F173L突变基因及D5正常基因转基因小鼠的血压和心脏结构与功能进行分析,了解多巴胺D5受体在高血压发生发展过程中的作用。方法利用无创血压测量仪和高分辨率小动物超声系统检测两种转基因小鼠的血压和左心室壁厚度、左心室内径、左心室容积、射血分数、短轴缩短率和左心室质量等心脏功能指标。结果 D5^F173L转基因小鼠4月龄、6月龄、16月龄时收缩压、舒张压都明显高于D5转基因小鼠;4月龄、6月龄的D5F173L转基因小鼠与D5转基因小鼠相比舒张期和收缩期左室壁厚度均明显增大、左室内容积均明显变小、左心室重量增加;16月龄的D5^F173L转基因小鼠与D5转基因小鼠相比左心室前壁增厚、心腔内径缩短,心腔容积下降、心室重量增加、射血分数提高、短轴缩短率提高;在18月龄时D5^F173L转基因小鼠相比于D5转基因小鼠左心室收缩期前壁厚度增加,后壁厚度减少,舒张期前壁厚度增加,后壁厚度减少;另外在18月龄时D5^F173L转基因小鼠与其16月龄时相比,射血分数、短轴缩短率明显降低,收缩期左心室容积明显增大。结论 D5^F173L转基因小鼠的血压及心脏功能与结构的分析结果符合原发性高血压的特征。D5^F173L转基因小鼠可作为原发性高血压动物模型。     

A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors

ATP-gated P2X4 receptors (P2X4R) are abundantly expressed in the CNS. However, little is known about the molecular targets for ethanol action in P2X4Rs. The current investigation tested the hypothesis that the ectodomain-transmembrane (TM) interface contains residues that are important for the action of ethanol in P2X4Rs. Wild type (WT) and mutant P2X4R were expressed in Xenopus oocytes. ATP concentration–response curves and ethanol (10–200 mM)-induced changes in ATP EC₁₀-gated currents were determined using two-electrode voltage clamp (−70 mV). Alanine substitution at the ectodomain-TM1 interface (positions 50–61) resulted in minimal changes in ethanol response. On the other hand, alanine substitution at the ectodomain-TM2 interface (positions 321–337) identified two key residues (D331 and M336) that significantly reduced ethanol inhibition of ATP-gated currents without causing marked changes in ATP I _max, EC₅₀, or Hill's slope. Other amino acid substitutions at positions 331 and 336 significantly altered or eliminated the modulatory effects of ethanol. Linear regression analyses revealed a significant relationship between hydropathy and polarity, but not molecular volume/molecular weight of the residues at these two positions. The results support the proposed hypothesis and represent an important step toward developing ethanol-insensitive receptors for investigating the role of P2X4Rs in mediating behavioral effects of ethanol.