Characterization of single channel currents from primary cilia of renal epithelial cells

The primary cilium is a ubiquitous, non-motile microtubular organelle lacking the central pair of microtubules found in motile cilia. Primary cilia are surrounded by a membrane, which has a unique complement of membrane proteins, and may thus be functionally different from the plasma membrane. The function of the primary cilium remains largely unknown. However, primary cilia have important sensory transducer properties, including the response of renal epithelial cells to fluid flow or mechanical stimulation. Recently, renal cystic diseases have been associated with dysfunctional ciliary proteins. Although the sensory properties of renal epithelial primary cilia may be associated with functional channel activity in the organelle, information in this regard is still lacking. This may be related to the inherent difficulties in assessing electrical activity in this rather small and narrow organelle. In the present study, we provide the first direct electrophysiological evidence for the presence of single channel currents from isolated primary cilia of LLC-PK1 renal epithelial cells. Several channel phenotypes were observed, and addition of vasopressin increased cation channel activity, which suggests the regulation, by the cAMP pathway of ciliary conductance. Ion channel reconstitution of ciliary versus plasma membranes indicated a much higher channel density in cilia. At least three channel proteins, polycystin-2, TRPC1, and interestingly, the alpha-epithelial sodium channel, were immunodetected in this organelle. Ion channel activity in the primary cilium of renal cells may be an important component of its role as a sensory transducer.     

Effect of unsaturated fatty acids in growth inhibition of some penicillin-resistant and sensitive bacteria

The growth of two penicillin-resistant Gram-positive bacteria, Bacillus licheniformis (749/C, penicillin G-resistant) and Staphylococcus aureus (metR 18, methicillin-resistant) and one Gram-negative strain, Escherichia coli (cloxacillin-resistant) as well as that of their wild counterparts was inhibited by the long-chain unsaturated fatty acids, linoleic, linolenic and arachidonic acid. The minimum inhibitory concentrations (MIC) of all the fatty acids were found to be 4–6 μg/ml for Staph. aureus (metR 18 & wild), 8–30 μg/ml for B. licheniformis (749/C & wild) and 70–90 μg/ml for E. coli (cloxacillin-resistant & wild). The inhibitory activity increased as the number of double bonds in the fatty acids increased. In most instances the concentrations of fatty acids required to inhibit the growth of the penicillin-resistant strains were lower than that required for their sensitive counterparts. This inhibition of growth in the presence of fatty acids may be due to an increase in permeability of the membrane as evidenced by the measurement of the leakage of 260 nm absorbing material and fluidity.     

Polycystin-2 cation channel function is under the control of microtubular structures in primary cilia of renal epithelial cells

Mutations in the gene encoding polycystin-2 (PC2) result in autosomal dominant polycystic kidney disease and defects in left-right asymmetry during embryogenesis. PC2 is a TRP-type Ca(2+)-permeable non-selective cation channel, which is expressed in kidney and other organs. PC2 is present and functional in microtubule-containing primary cilia of renal epithelial cells. However, no information is yet available as to whether PC2 interacts with microtubules. Here, we assessed the role of microtubular dynamics in regulating PC2 channel function in primary cilia. Isolated ciliary membranes from LLC-PK1 epithelial cells were reconstituted in a lipid bilayer system. The acute addition of the microtubular disrupter colchicine (15 mum) rapidly abolished, whereas the addition of the microtubular stabilizer paclitaxel (taxol, 15 mum) increased ciliary PC2 channel activity. The further addition of alpha-tubulin plus GTP also stimulated PC2 channel activity in ciliary membranes. However, alpha-tubulin and GTP had no effect on in vitro translated PC2. Using the yeast two-hybrid assay, we found that PC2 interacts with the microtubule-dependent motor kinesin-2 subunit KIF3A, a protein involved in polycystic kidney disease. The interaction occurred through the carboxyl termini domain of both proteins, which was further confirmed by in vitro glutathione S-transferase pull-down and dot blot overlay assays. Co-immunoprecipitation experiments showed that PC2 and KIF3A are in the same complex in native HEK293, Madin-Darby canine kidney cells (MDCK), and LLC-PK1 cells. Immunofluorescent staining also showed substantial PC2 and KIF3A co-localization in primary cilia of renal epithelial cells. The data indicate that microtubular organization regulates PC2 function, which may explain, among others, the regulatory role of PC2 in the sensory function of primary cilia.     

PACAP and gastrin regulate the histidine decarboxylase promoter via distinct mechanisms

The enterochromaffin-like (ECL) cell controls gastric acid secretion via histamine, generated by l-histidine decarboxylase (HDC). HDC expression is regulated by gastrin. However, gastrin is not alone in controlling ECL cell function. For example, the neural peptide pituitary adenylate cyclase-activating polypeptide (PACAP) also increases ECL cell proliferation. To investigate a potential role of PACAP in regulating HDC expression, we generated a series of HDC promoter-luciferase reporter constructs and transiently transfected them into PC12 cells (stably expressing the gastrin-CCK-2 receptor). We found that PACAP regulates HDC promoter activity. This is temporally biphasic, involving both adenyl cyclase and phospholipase C-dependent pathways. Deletional analysis, block mutation, and EMSA demonstrated a PACAP-response element at -177 to -170, wholly necessary for the effects of PACAP and discrete from known gastrin-responsive elements. Discrete neural and endocrine pathways regulate ECL cells through different patterns of postreceptor signaling and promoter activation, which may be appropriate to their functions in vivo.     

Gastrin regulates the heparin-binding epidermal-like growth factor promoter via a PKC/EGFR-dependent mechanism

Gastrin is a known growth/differentiation factor for the gastric mucosa. Its effects are likely mediated by the induction of heparin-binding epidermal-like growth factor (HB-EGF), a member of the EGF family of growth factors that is expressed by gastric parietal cells. In this study, we investigated the regulation of the HB-EGF promoter by gastrin in a human gastric cancer cell line. Serial human HB-EGF promoter-luciferase reporter deletion constructs and heterologous promoter constructs were transfected into AGS-E cells and stimulated with gastrin (10(-7) M) with or without various signal transduction inhibitors. EMSA were also performed. Gastrin stimulation resulted in a fivefold increase in HB-EGF-luciferase activity. The cis-acting element mediating gastrin responsiveness was mapped to the -69 to -58 region of the HB-EGF promoter. Gastrin stimulation was PKC dependent and at least partially mediated by activation of the EGF receptor.     

TCR mimic monoclonal antibodies induce apoptosis of tumor cells via immune effector-independent mechanisms

mAbs that recognize peptides presented on the cell surface by MHC class I molecules are potential therapeutic agents for cancer therapy. We have previously demonstrated that these Abs, which we termed TCR mimic mAbs (TCRm), reduce tumor growth in models of breast carcinoma. However, mechanisms of TCRm-mediated tumor growth reduction remain largely unknown. In this study, we report that these Abs, in contrast to several mAbs used currently in the clinic, destroy tumor cells independently of immune effector mechanisms such as Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We found that TCRm-mediated apoptosis of tumor cells was associated with selective and specific binding of these Abs to peptide/HLA class I complexes, which triggered the activation of JNK and intrinsic caspase pathways. This signaling was accompanied by the release of mitochondrial cytochrome c and apoptosis-inducing factor. TCRm-induced apoptosis in tumor cells was completely inhibited by soluble MHC tetramers loaded with relevant peptide as well as with inhibitors for JNK and caspases. Furthermore, mAbs targeting MHC class I, independent of the peptide bound by HLA, did not stimulate apoptosis, suggesting that the Ab-binding site on the MHC/peptide complex determines cytotoxicity. This study suggests the existence of mechanisms, in addition to ADCC and CDC, through which these therapeutic Abs destroy tumor cells. These mechanisms would appear to be of particular importance in severely immunocompromised patients with advanced neoplastic disease, since immune cell-mediated killing of tumor cells through ADCC and CDC is substantially limited in these individuals.     

Extranuclear DNA accumulates in aged cells and contributes to senescence and inflammation

Systemic inflammation is central to aging‐related conditions. However, the intrinsic factors that induce inflammation are not well understood. We previously identified a cell‐autonomous pathway through which damaged nuclear DNA is trafficked to the cytosol where it activates innate cytosolic DNA sensors that trigger inflammation. These results led us to hypothesize that DNA released after cumulative damage contributes to persistent inflammation in aging cells through a similar mechanism. Consistent with this notion, we found that older cells harbored higher levels of extranuclear DNA compared to younger cells. Extranuclear DNA was exported by a leptomycin B‐sensitive process, degraded through the autophagosome–lysosomal pathway and triggered innate immune responses through the DNA‐sensing cGAS‐STING pathway. Patient cells from the aging diseases ataxia and progeria also displayed extranuclear DNA accumulation, increased pIRF3 and pTBK1, and STING‐dependent p16 expression. Removing extranuclear DNA in old cells using DNASE2A reduced innate immune responses and senescence‐associated (SA) β‐gal enzyme activity. Cells and tissues of Dnase2a^?/? mice with defective DNA degradation exhibited slower growth, higher activity of β‐gal, or increased expression of HP‐1β and p16 proteins, while Dnase2a^?/?;Sting^?/? cells and tissues were rescued from these phenotypes, supporting a role for extranuclear DNA in senescence. We hypothesize a direct role for excess DNA in aging‐related inflammation and in replicative senescence, and propose DNA degradation as a therapeutic approach to remove intrinsic DNA and revert inflammation associated with aging.