Glucocorticoid-stimulated, transcription-independent release of annexin A1 by cochlear Hensen cells.

BACKGROUND AND PURPOSE: The current clinical strategy to protect the auditory organ against inflammatory damage by migrating leukocytes is the local delivery of glucocorticoids. However, the mechanism by which glucocorticoids confer this protection remains unknown. Therefore, we investigated the cellular and molecular targets of glucocorticoids in the cochlea that could be involved in preventing leukocyte migration. EXPERIMENTAL APPROACH: We used microscopy as well as immunocytochemical and microfluidic techniques to elucidate the effect of dexamethasone, hydrocortisone and prednisolone on the cellular and intracellular distribution of annexin A1 (ANXA1) - a glucocorticoid target known to inhibit leukocyte migration by receptor-mediated signalling - in the cochlea and isolated cochlear cells of guinea pigs. KEY RESULTS: All the cells lining the scala media - the cochlear compartment containing the auditory organ - express ANXA1 and the ANXA1 receptor FPR2/ALX is present in the scala media, as well as in other cochlear ducts. The majority of ANXA1 in the scala media is stored inside lipid droplets within cochlear Hensen cells. Glucocorticoids activate a myosin IIC-mediated mechanism that drives ANXA1 from the lipid droplets to the apical region of the Hensen cells, where ANXA1 is released to the external milieu by a process involving ABC transporters. CONCLUSIONS AND IMPLICATIONS: These findings suggest that ANXA1 could be a major mediator of the anti-inflammatory effects of glucocorticoids in the cochlea and identify new molecular targets for prevention of sudden sensorineural hearing loss.

Apoptosis in the OC-k3 immortalized cell line treated with different agents.

The aim of this study is to outline the mechanisms leading cochlear cells to die. We utilized an immortalized cell line (OC-k3 cells) derived from the organ of Corti of transgenic mice in order to perform in-depth biochemical studies with no limitations on sample size and number. We probed these cells with cisplatin and gentamicin, two drugs which display in vivo undesired ototoxic side-effects. We investigated cell viability, reactive oxygen species (ROS) production and glutathione (GSH) levels and tested the effects of different concentrations of cisplatin and gentamicin from 0 to 48 h. Results show that cells undergo a dose- and treatment-time-dependent apoptosis characterized by nuclear fragmentation, integrity of the cell membrane and mitochondria, and absence of DNA endonuclease activity. During the early part of treatment, ROS production increases and intracellular GSH decreases, probably due to the activation of protein kinase C alpha. Use of antioxidants such as N-acetylcysteine, GSH and vitamin C rescues cells from apoptosis almost completely. Overall, these data indicate that ROS generation might play a central role in inducing inner ear cell apoptosis and may have an additive role in the ageing process.

Rho GTPases mediate the regulation of cochlear outer hair cell motility by acetylcholine.

Outer hair cells are the mechanical effectors of the cochlear amplifier, an active process that improves the sensitivity and frequency discrimination of the mammalian ear. In vivo, the gain of the cochlear amplifier is regulated by the efferent neurotransmitter acetylcholine through the modulation of outer hair cell motility. Little is known, however, regarding the molecular mechanisms activated by acetylcholine. In this study, intracellular signaling pathways involving the small GTPases RhoA, Rac1, and Cdc42 have been identified as regulators of outer hair cell motility. Changes in cell length (slow motility) and in the amplitude of electrically induced movement (fast motility) were measured in isolated outer hair cells patch clamped in whole-cell mode, internally perfused through the patch pipette with different inhibitors and activators of these small GTPases while being externally stimulated with acetylcholine. We found that acetylcholine induces outer hair cell shortening and a simultaneous increase in the amplitude of fast motility through Rac1 and Cdc42 activation. In contrast, a RhoA- and Rac1-mediated signaling pathway induces outer hair cell elongation and decreases fast motility amplitude. These two opposing processes provide the basis for a regulatory mechanism of outer hair cell motility.

Establishment and characterization of conditionally immortalized organ of corti cell lines.

A culture of cells was isolated from the organ of Corti of 2-week-old H-2Kb-tsA58 (Immortomouse) transgenic mice. All cells of these mice harbor a mutant of the simian virus 40 A-gene, encoding a thermolabile large T-antigen (Tag) protein. At 33 degrees C the Tag protein is functional and induces cell proliferation, but at 39 degrees C it is rapidly denatured and inactivated. Isolated organ of Corti cells growing at 33 degrees C were predominantly small, rounded or fusiform and proliferated rapidly. When moved to 39 degrees C, the cells reduced their rate of proliferation and differentiated into specific morphological phenotypes. Four cell lines were cloned by limiting dilution and characterized by immunofluorescence microscopy and Western blot. The cell lines, named OC-k1, OC-k2, OC-k3 and OC-k4, have been passaged at least 50 times with retention of a stable phenotype. These cell lines were all positive for the neuroepithelial precursor cell marker nestin and for the inner ear cell marker OCP2. In addition, the cells showed reactivity to epithelial and neuronal cell markers, but with a pattern of protein expression different for each clone and different between cells of the same clone growing at 33 degrees C or 39 degrees C. Some of the clones exhibited asymmetric cell division which is a characteristic commonly ascribed to stem cells. These cell lines can be used advantageously to study mechanisms and signals involved in the control of cell differentiation and morphogenesis of the mammalian inner ear and to isolate inner ear specific proteins.

Localization of myosin-V in the centrosome.

The perinuclear localization of myosin-V was investigated in a variety of cultured mammalian cells and in primary cultures of rat hippocampus. In all cells investigated, myosin-V immunoreactivity was associated with the centrosome. In interphase cells, myosin-V was found in pericentriolar material, and in both mother and daughter centrioles. These results were obtained by using two different fixation protocols with three different affinity-purified antibodies that recognized a single band in Western blots. During cell division, myosin-V staining was intense throughout the cytoplasm and was concentrated in a trail between migrating centrioles and in the mitotic spindle poles and spindle fibers. The centrosome targeting site was determined to reside within the globular tail domain, because centrosome association also was observed in living cells transfected with DNA encoding the tail domain fused with a green fluorescent protein tag, but not in cells transfected with the vector encoding green fluorescent protein by itself.

A collagen substrate enhances the sealing capacity of tight junctions of A6 cell monolayers.

A6 cells, a kidney derived epithelial cell line, when cultured either on a collagen-coated substrate or on polycarbonate substrate without collagen form confluent monolayers that are similar in cell density and overall morphology. However, the transepithelial electrical resistance (TER) of monolayers grown on the collagen-coated substrate is ninefold higher than that of monolayers grown without collagen. A comparative freeze-fracture study showed that this large difference in TER is not related to the length or number of tight junction strands but to differences in the specific conductance of individual strands. This conductance was obtained considering the TER, the linear junctional density and the mean number of tight junction strands. We estimated the specific linear conductance of the tight junction strands to be 2.56 x 10(-7) S/cm for cells grown on collagen and 30.3 x 10(-7) S/cm for the cells grown without collagen. We also examined changes in distribution and phosphorylation states of the zonula occludens associated protein, ZO-1, during monolayer formation. Immunocytochemistry reveals that the distribution of ZO-1 follows a similar time course and pattern independent of the presence or absence of collagen. While the amount of ZO-1 expression is identical in cells grown on both substrates, this protein is phosphorylated to a greater extent during the initial stages of confluence in cells cultured on collagen. We suggest that the phosphorylation levels of ZO-1 in A6 cells at the early stages of monolayer formation may determine the final molecular structure and specific conductance of the tight junctions strands.

Immunolocalization of anion exchanger 2alpha in auditory sensory hair cells.

We have previously reported the isolation from a guinea pig organ of Corti cDNA library of a cDNA clone that encodes a novel isoform of the anion exchanger 2 (AE2) protein (Negrini, Rivolta, Kalinec and Kachar, 1995. Cloning of an organ of Corti anion exchanger 2 isoform with a truncated C-terminal domain. Biophys. Acta, 1236, 207-211). The deduced protein, named AE2alpha, has a conserved cytoplasmic domain and a short membrane domain with only two membrane spanning regions, as opposed to the fourteen present in the conventional AE2. Now, we are showing the immunolocalization and preliminary characterization of this protein using an antipeptide antibody specific for this novel AE2 isoform. In Western blots, this antibody binds to an approximately 89 kDa polypeptide that corresponds to a phosphorylated protein with serines as main phosphate acceptor residues. In immunofluorescence experiments, the antibody labels the stereocilia and the lateral wall of the outer hair cells and the stereocilia of the inner hair cells. Our results suggest that AE2alpha is a membrane-cytoskeletal linker in regions of the hair cell, where sensory transduction mechanisms take place.

Transforming G protein-coupled receptors potently activate JNK (SAPK). Evidence for a divergence from the tyrosine kinase signaling pathway.

The expression of human muscarinic acetylcholine receptors (mAChRs) in NIH 3T3 cells has been used as a model for studying proliferative signaling through G protein-coupled receptors. In this biological system, the m1 class of mAChRs can effectively transduce mitogenic signals (Stephens, E.V., Kalinec, G., Brann, M.R., and Gutkind, J.S. (1993) Oncogene 8, 19-26) and induce malignant transformation if persistently activated (Gutkind, J.S., Novotny, E.A., Brann, M.R., and Robbins, K.C. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4703-4708). Moreover, available evidence suggests that the m1-signaling pathway converges at the level of p21ras with that emerging from tyrosine kinase receptors (Crespo, P., Xu, N., Simonds, W.F., and Gutkind, J.S. (1994) Nature 369, 418-420). To explore nuclear events involved in growth regulation by G protein-coupled receptors in this setting, we compared the effect of platelet-derived growth factor (PDGF) and the cholinergic agonist, carbachol, on the expression of mRNA for members of the jun and fos family of nuclear proto-oncogenes. We found that activation of m1 receptors by carbachol induces the expression of a distinct set of nuclear transcription factors. In particular, carbachol caused a much greater induction of c-jun mRNA and AP-1 activity. These responses did not correlate with protein kinase C stimulation nor with the activation of mitogen-activated protein (MAP) kinases. Recently, it has been shown that a novel family of kinases structurally related to MAP kinases, stress-activated protein kinases, or Jun kinases (JNKs), phosphorylate in vivo the amino-terminal transactivating domain of the c-Jun protein, thereby increasing its transcriptional activity. In view of our results, this observation prompted us to ask whether m1 and PDGF can differentially activate JNKs. Here, we show that m1 mAChRs can induce a remarkable increase in JNK activity, which was temporally distinct from that of MAP kinase and was entirely protein kinase C independent. In contrast, PDGF failed to activate JNK in these cells, although it stimulated MAP kinase to an extent even greater than that for carbachol. These findings demonstrate that G protein-coupled receptors can signal through pathways leading to the activation of JNK, thus diverging at this level with those signaling routes utilized by tyrosine kinase receptors.

Transforming G protein-coupled receptors transduce potent mitogenic signals in NIH 3T3 cells independent on cAMP inhibition or conventional protein kinase C.

We have used the expression of human acetylcholine muscarinic receptor (mAChR) genes in NIH 3T3 cells as a model for dissecting the molecular basis of cellular transformation induced by G protein-coupled receptors. Those mAChR subtypes efficiently coupled to PIP2 hydrolysis (m1, m3 and m5) induced agonist-dependent cell transformation whereas those inhibiting adenylyl cyclase (m2, m4) lack transforming activity. In the present study, we demonstrate that in cells expressing m1 but not m2 mAChRs the cholinergic agonist (carbachol) is alone as potent a stimulant for DNA synthesis as platelet-derived growth factor (PDGF) or serum. Furthermore, induction of DNA synthesis is shown to correlate with activation of PIP2 hydrolysis but not with inhibition of adenylyl cyclase. We also examined the role of protein kinase C (PKC) in mitogenic signalling through m1 mAChRs, and found that NIH 3T3 cells express PKC-alpha and PCK-zeta as the only conventional or Ca(2+)-independent PKC isozyme, respectively. Prolonged treatment with TPA depleted cells of PKC-alpha but not of PKC-zeta. In TPA-treated NIH 3T3 cells, the mitogenic response to a subsequent stimulation with TPA was absolutely abolished, but the response to PDGF or serum was not. Moreover, PKC depletion did not decrease DNA synthesis induced by carbachol. We conclude that carbachol potently induces reinitiation of DNA synthesis through the activation of transforming mAChR subtypes, independently of inhibition of adenylyl cyclase and conventional PKCs.

Mutated alpha subunit of the Gq protein induces malignant transformation in NIH 3T3 cells.

The discovery of mutated, GTPase-deficient alpha subunits of Gs or Gi2 in certain human endocrine tumors has suggested that heterotrimeric G proteins play a role in the oncogenic process. Expression of these altered forms of G alpha s or G alpha i2 proteins in rodent fibroblasts activates or inhibits endogenous adenylyl cyclase, respectively, and causes certain alterations in cell growth. However, it is not clear whether growth abnormalities result from altered cyclic AMP synthesis. In the present study, we asked whether a recently discovered family of G proteins, Gq, which does not affect adenylyl cyclase activity, but instead mediates the activation of phosphatidylinositol-specific phospholipase C harbors transforming potential. We mutated the cDNA for the alpha subunit of murine Gq in codons corresponding to a region involved in binding and hydrolysis of GTP. Similar mutations unmask the transforming potential of p21ras or activate the alpha subunits of Gs or Gi2. Our results show that when expressed in NIH 3T3 cells, activating mutations convert G alpha q into a dominant acting oncogene.