Recent findings show that cilia are sensory organelles that display specific receptors and ion channels, which transmit signals from the extracellular environment via the cilium to the cell to control tissue homeostasis and function. Agenesis of primary cilia or mislocation of ciliary signal components affects human pathologies, such as polycystic kidney disease and disorders associated with Bardet-Biedl syndrome. Primary cilia are essential for hedgehog ligand-induced signaling cascade regulating growth and patterning. Here, we show that the primary cilium in fibroblasts plays a critical role in growth control via platelet-derived growth factor receptor alpha (PDGFRalpha), which localizes to the primary cilium during growth arrest in NIH3T3 cells and primary cultures of mouse embryonic fibroblasts. Ligand-dependent activation of PDGFRalphaalpha is followed by activation of Akt and the Mek1/2-Erk1/2 pathways, with Mek1/2 being phosphorylated within the cilium and at the basal body. Fibroblasts derived from Tg737(orpk) mutants fail to form normal cilia and to upregulate the level of PDGFRalpha; PDGF-AA fails to activate PDGFRalphaalpha and the Mek1/2-Erk1/2 pathway. Signaling through PDGFRbeta, which localizes to the plasma membrane, is maintained at comparable levels in wild-type and mutant cells. We propose that ciliary PDGFRalphaalpha signaling is linked to tissue homeostasis and to mitogenic signaling pathways.

The effects of gonadotropins on progesterone receptor (PR) expression and localization in the mouse oviduct, uterus, and ovary was examined. In the oviduct ciliated epithelial cells of adult mice and human revealed a unique PR localization to the lower half of the motile cilia whereas the nuclei were unstained or faintly stained. Pubertal female mice were further studied by confocal laser scanning microscopy and western blotting before and after injection with FSH and LH followed by human chorionic gonadotropin (hCG) injection after a 48-h period. PR immunolocalization to the oviduct cilia was greatly increased in pubertal mice upon hCG stimulation. In neighboring goblet cells, the PR staining was confined to the nuclei. Nuclear PR localization was evident in epithelial cells of the uterus as well as in a fraction of stromal and muscle cells. Staining intensity and number of stained cells was not affected by hormone stimulation. In the ovary, weak PR immunolocalization was observed in unprimed animals but increased significantly after hCG stimulation. In granulosa cells of preovulatory follicles PR was exclusively observed in mural cells, whereas cumulus cells remained negative. At all stages examined, primary granulosa cell cilia lacked PR staining. SDS-PAGE and western blotting analysis of tissues from oviduct, uterus, and ovary confirmed antibody specificity, and identified two bands corresponding to the PR isoforms PR-A and PR-B. Upon hCG stimulation, a new band cross-reacting with anti-PR emerged above the PR-A form in oviduct fractions, suggesting LH-induced phosphorylation of PR-A. We suggest that ciliary PR in the oviduct plays a role in progesterone signaling after ovulation, possibly via non-genomic events. These novel findings warrant further studies of oviduct and postovulatory signaling events and suggest a sensory role for oviduct cilia in the process of oocyte transport/fertilization.

The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.Connexin43 (Cx43) 1 -mediated gap junctional communication regulates bone matrix production in vitro (1) and is required for normal bone formation in vivo (2). Modulation of gap junctional communication in vitro alters osteocalcin production by transcriptional regulation. In vivo, detailed analysis of Cx43-deficient mice has revealed distinctive skeletal abnormalities. However, the mechanisms by which Cx43-mediated gap junctional communication alter bone formation are poorly understood. One such mechanism may be by the coordination of intercellular calcium signaling among osteoblastic cells. A well described in vitro model, and one which may mimic the effects of mechanical forces on bone cells, is that of mechanically induced intercellular calcium signaling or intercellular calcium waves (ICW).Two mechanisms for the propagation of mechanically induced ICW in osteoblastic cells have been described. In the UMR 106 -01 rat osteoblastic cell line, mechanical stimulation of a single osteoblast leads to propagation of fast (ϳ15 m/s) ICW that require activation of P2Y (purinergic) receptors on neighboring cells, presumably by released nucleotides, and subsequent generation of inositol trisphosphate and release of intracellular calcium stores. In the ROS 17/2.5 rat osteoblastic cell line, slow ICW require gap junctional communication. Unlike gap junction-mediated ICW that have been identified in many other cell models, gap junction-mediated ICW in ROS cells do not require release of intracellular calcium stores and do not require diffusion of IP 3 through gap junction pores. In human osteoblastic cells, both of these mechanisms for the propagation of ICW have been identified and have different kinetics. Mechanical stimulation of human osteoblast-like cells results in fast calcium waves that require activation of P2Y receptors. If these waves are blocked by desensitization of P2Y receptors, slow...

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We have examined the subcellular localization of transient receptor potential (TRP) ion channels and the potential sensory role of cilia in murine female reproductive organs using confocal laser scanning microscopy analysis on ovary and oviduct tissue sections as well as on primary cultures of follicular granulosa cells. We show that the Ca2+ permeable cation channel, polycystin-2, as well as polycystin-1, a receptor that forms a functional protein complex with polycystin 2, distinctively localize to primary cilia emerging from granulosa cells of antral follicles in vivo and in vitro. Both polycystins are localized to motile oviduct cilia and this localization is greatly increased upon ovulatory gonadotropic stimulation. Further, the Ca2+ permeable cation channel, TRP vaniloid 4 (TRPV4), localizes to a sub-population of motile cilia on the epithelial cells of the ampulla and isthmus with high intensity in proximal invaginations of the epithelial folds. These observations are the first to demonstrate ciliary localization of TRP ion channels and their possible receptor function in the female reproductive organs. We suggest that polycystins 1 and 2 play an important role in granulosa cell differentiation and in development and maturation of ovarian follicles. In the oviduct both TRPV4 and polycystins could be important in relaying physiochemical changes in the oviduct upon ovulation.