Ye Chun Ruan

The cystic fibrosis transmembrane conductance regulator in reproductive health and disease

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel regulated by cAMP‐dependent phosphorylation, which is expressed in epithelial cells of a wide variety of tissues including the reproductive tracts. Mutations in the gene encoding CFTR cause cystic fibrosis, a common genetic disease in Caucasian populations with a multitude of clinical manifestations including infertility/subfertility in both sexes. However, the physiological role of CFTR in reproduction and its involvement in the pathogenesis of reproductive diseases remain largely unknown. This review discusses the role of CFTR in regulating fluid volume and bicarbonate secretion in the reproductive tracts and their importance in various reproductive events. We also discuss the contribution of CFTR dysfunction to a number of pathological conditions. The evidence presented is consistent with an important role of CFTR in reproductive health and disease, suggesting that CFTR might be a potential target for the diagnosis and treatment of reproductive diseases including infertility.

Implant-Derived Magnesium Induces Local Neuronal Production of CGRP to Improve Bone-Fracture Healing in Rats

Orthopedic implants containing biodegradable magnesium have been used for fracture repair with considerable efficacy; however, the underlying mechanisms by which these implants improve fracture healing remain elusive. Here we show the formation of abundant new bone at peripheral cortical sites after intramedullary implantation of a pin containing ultrapure magnesium into the intact distal femur in rats. This response was accompanied by substantial increases of neuronal calcitonin gene-related polypeptide-α (CGRP) in both the peripheral cortex of the femur and the ipsilateral dorsal root ganglia (DRG). Surgical removal of the periosteum, capsaicin denervation of sensory nerves or knockdown in vivo of the CGRP-receptor-encoding genes Calcrl or Ramp1 substantially reversed the magnesium-induced osteogenesis that we observed in this model. Overexpression of these genes, however, enhanced magnesium-induced osteogenesis. We further found that an elevation of extracellular magnesium induces magnesium transporter 1 (MAGT1)-dependent and transient receptor potential cation channel, subfamily M, member 7 (TRPM7)-dependent magnesium entry, as well as an increase in intracellular adenosine triphosphate (ATP) and the accumulation of terminal synaptic vesicles in isolated rat DRG neurons. In isolated rat periosteum-derived stem cells, CGRP induces CALCRL- and RAMP1-dependent activation of cAMP-responsive element binding protein 1 (CREB1) and SP7 (also known as osterix), and thus enhances osteogenic differentiation of these stem cells. Furthermore, we have developed an innovative, magnesium-containing intramedullary nail that facilitates femur fracture repair in rats with ovariectomy-induced osteoporosis. Taken together, these findings reveal a previously undefined role of magnesium in promoting CGRP-mediated osteogenic differentiation, which suggests the therapeutic potential of this ion in orthopedics.

Activation of the epithelial Na + channel triggers prostaglandin E 2 release and production required for embryo implantation

Embryo implantation remains a poorly understood process. We demonstrate here that activation of the epithelial Na+ channel (ENaC) in mouse endometrial epithelial cells by an embryo-released serine protease, trypsin, triggers Ca2+ influx that leads to prostaglandin E2 (PGE2) release, phosphorylation of the transcription factor CREB and upregulation of cyclooxygenase 2, the enzyme required for prostaglandin production and implantation. We detected maximum ENaC activation, as indicated by ENaC cleavage, at the time of implantation in mice. Blocking or knocking down uterine ENaC in mice resulted in implantation failure. Furthermore, we found that uterine ENaC expression before in vitro fertilization (IVF) treatment is markedly lower in women with implantation failure as compared to those with successful pregnancy. These results indicate a previously undefined role of ENaC in regulating the PGE2 production and release required for embryo implantation, defects that may be a cause of miscarriage and low success rates in IVF.

Glucose-induced electrical activities and insulin secretion in pancreatic islet β-cells are modulated by CFTR

The cause of insulin insufficiency remains unknown in many diabetic cases. Up to 50% adult patients with cystic fibrosis (CF), a disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), develop CF-related diabetes (CFRD) with most patients exhibiting insulin insufficiency. Here we show that CFTR is a regulator of glucose-dependent electrical acitivities and insulin secretion in β-cells. We demonstrate that glucose elicited whole-cell currents, membrane depolarization, electrical bursts or action potentials, Ca2+ oscillations and insulin secretion are abolished or reduced by inhibitors or knockdown of CFTR in primary mouse β-cells or RINm5F β-cell line, or significantly attenuated in CFTR mutant (DF508) mice compared with wild-type mice. VX-809, a newly discovered corrector of DF508 mutation, successfully rescues the defects in DF508 β-cells. Our results reveal a role of CFTR in glucose-induced electrical activities and insulin secretion in β-cells, shed light on the pathogenesis of CFRD and possibly other idiopathic diabetes, and present a potential treatment strategy.

Regulation of smooth muscle contractility by the epithelium in rat vas deferens: role of ATP-induced release of PGE2

Recent studies suggest that the epithelium might modulate the contractility of smooth muscle. However, the mechanisms underlying this regulation are unknown. The present study investigated the regulation of smooth muscle contraction by the epithelium in rat vas deferens and the possible factor(s) involved. Exogenously applied ATP inhibited electrical field stimulation (EFS)‐evoked smooth muscle contraction in an epithelium‐dependent manner. As the effects of ATP on smooth muscle contractility were abrogated by inhibitors of prostaglandin synthesis, but not by those of nitric oxide synthesis, prostaglandins might mediate the effects of ATP. Consistent with this idea, PGE2 inhibited EFS‐evoked smooth muscle contraction independent of the epithelium, while ATP and UTP induced the release of PGE2 from cultured rat vas deferens epithelial cells, but not smooth muscle cells. The ATP‐induced PGE2 release from vas deferens epithelial cells was abolished by U73122, an inhibitor of phospholipase C (PLC) and BAPTA AM, a Ca2+ chelator. ATP also transiently increased [Ca2+]i in vas deferens epithelial cells. This effect of ATP on [Ca2+]i was independent of extracellular Ca2+, but abolished by the P2 receptor antagonist RB2 and U73122. In membrane potential measurements using a voltage‐sensitive dye, PGE2, but not ATP, hyperpolarized vas deferens smooth muscle cells and this effect of PGE2 was blocked by MDL12330A, an adenylate cyclase inhibitor, and the chromanol 293B, a blocker of cAMP‐dependent K+ channels. Taken together, our results suggest that ATP inhibition of vas deferens smooth muscle contraction is epithelium dependent. The data also suggest that ATP activates P2Y receptor‐coupled Ca2+ mobilization leading to the release of PGE2 from epithelial cells, which in turn activates cAMP‐dependent K+ channels in smooth muscle cells leading to the hyperpolarization of membrane voltage and the inhibition of vas deferens contraction. Thus, the present findings suggest a novel regulatory mechanism by which the epithelium regulates the contractility of smooth muscle.

Cellular Mechanisms Underlying the Laxative Effect of Flavonol Naringenin on Rat Constipation Model

Background & Aims Symptoms of constipation are extremely common, especially in the elderly. The present study aim to identify an efficacious treatment strategy for constipation by evaluating the secretion-promoting and laxative effect of a herbal compound, naringenin, on intestinal epithelial anion secretion and a rat constipation model, respectively. Methods/Principal Findings In isolated rat colonic crypts, mucosal addition of naringenin (100 µM) elicited a concentration-dependent and sustained increase in the short-circuit current (ISC), which could be inhibited in Cl− free solution or by bumetanide and DPC (diphenylamine-2-carboxylic acid), but not by DIDS (4, 4′- diisothiocyanatostilbene-2, 2′-disulfonic acid). Naringenin could increase intracellular cAMP content and PKA activity, consisted with that MDL-12330A (N-(Cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride) pretreatment reduced the naringenin-induced ISC. In addition, significant inhibition of the naringenin-induced ISC by quinidine indicated that basolateral K+ channels were involved in maintaining this cAMP-dependent Cl− secretion. Naringenin-evoked whole cell current which exhibited a linear I–V relationship and time-and voltage- independent characteristics was inhibited by DPC, indicating that the cAMP activated Cl− conductance most likely CFTR (cystic fibrosis transmembrane conductance regulator) was involved. In rat constipation model, administration of naringenin restored the level of fecal output, water content and mucus secretion compared to loperamide-administrated group. Conclusions Taken together, our data suggest that naringenin could stimulate Cl− secretion in colonic epithelium via a signaling pathway involving cAMP and PKA, hence provide an osmotic force for subsequent colonic fluid secretion by which the laxative effect observed in the rat constipation model. Naringenin appears to be a novel alternative treatment strategy for constipation.

cSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit

Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associated with capacitation. We report here that, despite normal phosphorylation, sperm from cSrc null mice display a severe reduction in forward motility, and are unable to fertilize in vitro. Histological analysis of seminiferous tubules in the testes, caput and corpus epididymis do not reveal obvious defects. However, the cauda epididymis is significantly smaller, and expression of key transport proteins in the epithelial cells lining this region is reduced in cSrc null mice compared to wild type littermates. Although previously, we and others have shown the presence of cSrc in mature sperm from cauda epididymis, a closer evaluation indicates that this tyrosine kinase is not present in sperm from the caput epididymis, suggesting that this protein is acquired by sperm later during epididymal maturation. Consistent with this observation, cSrc is enriched in vesicles released by the epididymal epithelium known as epididymosomes. Altogether, these observations indicate that cSrc is essential for cauda epididymal development and suggest an essential role of this kinase in epididymal sperm maturation involving cSrc extracellular trafficking.

Regulation of Smooth Muscle Contraction by the Epithelium: Role of Prostaglandins

As an analog to the endothelium situated next to the vascular smooth muscle, the epithelium is emerging as an important regulator of smooth muscle contraction in many vital organs/tissues by interacting with other cell types and releasing epithelium-derived factors, among which prostaglandins have been demonstrated to play a versatile role in governing smooth muscle contraction essential to the physiological and pathophysiological processes in a wide range of organ systems.

ATP secretion in the male reproductive tract: essential role of CFTR

CFTR is expressed in principal cells but not clear cells in mouse cauda epididymis. Inhibition or knockdown of CFTR inhibits ATP release from mouse epididymal principal cells. Inhibition of CFTR reduces ATP release into the lumen of cauda epididymis in mice in vivo. These results show the involvement of CFTR in the regulation of ATP release from epithelial principal cells in the cauda epididymidis. Defective ATP signalling in the epididymis might contribute to dysfunction of the male reproductive tract associated with CFTR mutations.

Dedifferentiation-Reprogrammed Mesenchymal Stem Cells with Improved Therapeutic Potential†‡§

Stem cell transplantation has been shown to improve functional outcome in degenerative and ischemic disorders. However, low in vivo survival and differentiation potential of the transplanted cells limits their overall effectiveness and thus clinical usage. Here we show that, after in vitro induction of neuronal differentiation and dedifferentiation, on withdrawal of extrinsic factors, mesenchymal stem cells (MSCs) derived from bone marrow, which have already committed to neuronal lineage, revert to a primitive cell population (dedifferentiated MSCs) retaining stem cell characteristics but exhibiting a reprogrammed phenotype distinct from their original counterparts. Of therapeutic interest, the dedifferentiated MSCs exhibited enhanced cell survival and higher efficacy in neuronal differentiation compared to unmanipulated MSCs both in vitro and in vivo, with significantly improved cognition function in a neonatal hypoxic–ischemic brain damage rat model. Increased expression of bcl‐2 family proteins and microRNA‐34a appears to be the important mechanism giving rise to this previously undefined stem cell population that may provide a novel treatment strategy with improved therapeutic efficacy. STEM CELLS 2011;29:2077–2089.