Recruitment of endosomal signaling mediates the forskolin modulation of guinea pig cardiac neuron excitability.

Forskolin, a selective activator of adenylyl cyclase (AC), commonly is used to establish actions of G protein-coupled receptors (GPCRs) that are initiated primarily through activation of AC/cAMP signaling pathways. In the present study, forskolin was used to evaluate the potential role of AC/cAMP, which is a major signaling mechanism for the pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor, in the regulation of guinea pig cardiac neuronal excitability. Forskolin (5-10 µM) increases excitability in ~60% of the cardiac neurons. The forskolin-mediated increase in excitability was considered related to cAMP regulation of a cyclic nucleotide gated channel or via protein kinase A (PKA)/ERK signaling, mechanisms that have been linked to PAC1 receptor activation. However, unlike PACAP mechanisms, forskolin enhancement of excitability was not significantly reduced by treatment with cesium to block currents through hyperpolarization-activated nonselective cation channels (Ih) or by treatment with PD98059 to block MEK/ERK signaling. In contrast, treatment with the clathrin inhibitor Pitstop2 or the dynamin inhibitor dynasore eliminated the forskolin-induced increase in excitability; treatments with the inactive Pitstop analog or PP2 treatment to inhibit Src-mediated endocytosis mechanisms were ineffective. The PKA inhibitor KT5702 significantly suppressed the forskolin-induced change in excitability; further, KT5702 and Pitstop2 reduced the forskolin-stimulated MEK/ERK activation in cardiac neurons. Collectively, the present results suggest that forskolin activation of AC/cAMP/PKA signaling leads to the recruitment of clathrin/dynamin-dependent endosomal transduction cascades, including MEK/ERK signaling, and that endosomal signaling is the critical mechanism underlying the forskolin-induced increase in cardiac neuron excitability.

Differential Regulation of Clathrin and Its Adaptor Proteins during Membrane Recruitment for Endocytosis.

In plants, clathrin-mediated endocytosis (CME) is dependent on the function of clathrin and its accessory heterooligomeric adaptor protein complexes, ADAPTOR PROTEIN2 (AP-2) and the TPLATE complex (TPC), and is negatively regulated by the hormones auxin and salicylic acid (SA). The details for how clathrin and its adaptor complexes are recruited to the plasma membrane (PM) to regulate CME, however, are poorly understood. We found that SA and the pharmacological CME inhibitor tyrphostin A23 reduce the membrane association of clathrin and AP-2, but not that of the TPC, whereas auxin solely affected clathrin membrane association, in Arabidopsis (Arabidopsis thaliana). Genetic and pharmacological experiments revealed that loss of AP2µ or AP2σ partially affected the membrane association of other AP-2 subunits and that the AP-2 subunit AP2σ, but not AP2µ, was required for SA- and tyrphostin A23-dependent inhibition of CME Furthermore, we show that although AP-2 and the TPC are both required for the PM recruitment of clathrin in wild-type cells, the TPC is necessary for clathrin PM association in AP-2-deficient cells. These results indicate that developmental signals may differentially modulate the membrane recruitment of clathrin and its core accessory complexes to regulate the process of CME in plant cells.

An Aß42 uptake and degradation via Rg3 requires an activation of caveolin, clathrin and Aß-degrading enzymes in microglia.

We demonstrated previously that ginsenoside Rg3 enhances the expression of macrophage scavenger receptor class A (SRA) and amyloid ß peptide 1-42 (Aß42) uptake in BV2 cells. In this study, we investigated the biochemical and mechanistic roles of Rg3 in human microglia and animal models to identify the determinants that participate in restoring memory and learning in brains disrupted by the Aß42 peptide. SRA was expressed highly in Rg3-treated rats, and learning and memory functions were maintained at a normal level after the infusion of Aß42. SRA-transfected HMO6 human microglial cells (HMO6.hSRA) overexpressed SRA and took up a remarkable amount of Aß42. Rg3-treated HMO6 cells showed highly enhanced SRA expression and dramatically promoted Aß42 uptake. Moreover, high levels of clathrin and caveolin1 supported the roles of Rg3 in endocytic biogenesis by activating p38 and extracellular signal-regulated protein kinase signaling. Notably, both neprilysin (NEP) and insulin-degrading enzyme (IDE) were significantly expressed by Rg3, suggesting independent and compensatory hydrolytic activity for the Aß peptide. In conclusion, Rg3 successfully triggered Aß42 uptake via SRA and clathrin-/caveolae-mediated endocytic mechanisms and further contributed to accelerate the degradation of Aß peptide via the increase of intracellular NEP and IDE, which may be a promising Alzheimer׳s disease therapy.

Mechanistic studies on the uptake and intracellular trafficking of novel cyclodextrin transfection complexes by intestinal epithelial cells.

Oral delivery of gene therapeutics would facilitate treatment of local intestinal disease, including colon cancer and inflammatory bowel disease, thus avoiding invasive surgery. The aims of this study were to investigate; if the orientation of the lipid tail on the cyclodextrin (CD) influenced the efficacy of a novel poly-6-cationic amphiphilic CD to transfect intestinal enterocytes; the endocytotic uptake pathway(s), and, the intracellular trafficking of the CD·DNA complexes. Inhibitors of clathrin- and caveolae-mediated endocytosis and macropinocytosis were used to determine the mechanism(s) of CD·DNA uptake by both undifferentiated and differentiated Caco-2 cells. Cell surface heparan sulphate proteoglycans were involved in the association of CD·DNA complexes with undifferentiated Caco-2 cells. Complexation of pDNA with CD facilitated significant levels of pDNA uptake and gene expression (comparable to PEI) in both undifferentiated and differentiated Caco-2 cells. Disruption of intracellular vesicular trafficking reduced transfection activity. CD was also capable of transfecting the more physiologically relevant differentiated Caco-2 model. Macropinocytosis was responsible for the uptake of CD·DNA transfection complexes by both undifferentiated and differentiated Caco-2 cells. The ability of this novel CD to transfect differentiated intestinal cells indicates the potential of this vector for oral gene delivery.

Epidermal growth factor promotes epidermal growth factor receptor nuclear accumulation by a pathway dependent on cytoskeleton integrity in human breast cancer cells.

BACKGROUND AND AIMS: The epidermal growth factor receptor (EGFR) is activated by extracellular ligands of the epidermal growth factor (EGF) family, resulting in a cascade of cytoplasmic signaling events. Emerging evidence indicates a mode of EGF signaling in which growth factor signals are transmitted via EGFR nuclear transport. The aim of this study was to determine whether EGF promotes EGFR nuclear accumulation and the role of clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity in breast cancer cells. METHODS: MCF-7 cells were treated without or with 100 ng/ml EGF for various times and nuclear extracts were obtained. Nuclear accumulation of EGFR was analyzed by SDS-PAGE followed by Western blotting of nuclear extracts using an anti-EGFR Ab or with a phosphospecific Ab against the Tyr-1068 of EGFR and with anti-Rb Ab as the loading control. DNA binding activity of EGFR was analyzed by EMSA using nuclear extracts and a radiolabeled oligonucleotide probe representing the AT-rich minimal sequence (ATRS). RESULTS: EGF induces the nuclear accumulation of EGFR, an increase in EGFR phosphorylation at Tyr-1068 and the formation of the complex EGFR-DNA in MCF-7 and MDA-MB-231 breast cancer cells. In addition, EGFR nuclear accumulation is dependent of clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity. CONCLUSIONS: This study demonstrates that in breast cancer cells EGF promotes nuclear accumulation of EGFR and is dependent on clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity.

On the mechanism of internalization of alpha-synuclein into microglia: roles of ganglioside GM1 and lipid raft.

ALpha-synuclein (alpha-syn) has been known to be a key player of the pathogenesis of Parkinson's disease and has recently been detected in extracellular biological fluids and shown to be rapidly secreted from cells. The penetration of alpha-syn into cells has also been observed. In this study, we observed that dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a glucosyltransferase inhibitor, and proteinase K inhibited the internalization of extracellular monomeric alpha-syn into BV-2 cells, and the addition of monosialoganglioside GM1 ameliorated the inhibition of alpha-syn internalization in dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol-treated BV-2 cells. Furthermore, inhibition of clathrin-, caveolae-, and dynamin-dependent endocytosis did not prevent the internalization of alpha-syn, but disruption of lipid raft inhibited it. Inhibition of macropinocytosis and disruption of actin and microtubule structures also did not inhibit the internalization of alpha-syn. In addition, we further confirmed these observations by co-culture system of BV-2 cells and alpha-syn-over-expressing SH-SY5Y cells. These findings suggest that extracellular alpha-syn is internalized into microglia via GM1 as well as hitherto-unknown protein receptors in clathrin-, caveolae-, and dynamin-independent, but lipid raft-dependent manner. Elucidation of the mechanism involved in internalization of alpha-syn should be greatly helpful in the development of new treatments of alpha-syn-related neurodegenerative diseases.

Kaposi's sarcoma-associated herpesvirus utilizes an actin polymerization-dependent macropinocytic pathway to enter human dermal microvascular endothelial and human umbilical vein endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes clathrin-mediated endocytosis for its infectious entry into human foreskin fibroblast (HFF) cells (S. M. Akula, P. P. Naranatt, N.-S. Walia, F.-Z. Wang, B. Fegley, and B. Chandran, J. Virol. 77:7978-7990, 2003). Here, we characterized KSHV entry into primary human microvascular dermal endothelial (HMVEC-d) and human umbilical vein endothelial (HUVEC) cells. Similar to the results for HMVEC-d cells, KSHV infection of HUVEC cells also resulted in an initial high level and subsequent decline in the expression of the lytic switch gene, ORF50, while latent gene expression persisted. Internalized virus particles enclosed in irregular vesicles were observed by electron microscopy of infected HMVEC-d cells. At an early time of infection, colocalization of KSHV capsid with envelope was observed by immunofluorescence analysis, thus demonstrating endocytosis of intact enveloped virus particles. Chlorpromazine, an inhibitor of clathrin-mediated endocytosis, and filipin (C(35)H(58)O(11)), a caveolar endocytosis inhibitor, did not have any effect on KSHV binding, entry (DNA internalization), or gene expression in HMVEC-d and HUVEC cells. In contrast to the results for HFF cells, virus entry and gene expression in both types of endothelial cells were significantly blocked by macropinocytosis inhibitors (EIPA [5-N-ethyl-N-isoproamiloride] and rottlerin [C(30)H(28)O(8)]) and by cytochalasin D, which affects actin polymerization. Inhibition of lipid raft blocked viral gene expression in HMVEC-d cells but not in HUVEC or HFF cells. In HMVEC-d and HUVEC cells, KSHV induced the actin polymerization and formation of lamellipodial extensions that are essential for macropinocytosis. Inhibition of macropinocytosis resulted in the distribution of viral capsids at the HMVEC-d cell periphery, and capsids did not associate with microtubules involved in the nuclear delivery of viral DNA. Internalized KSHV in HMVEC-d and HUVEC cells colocalized with the macropinocytosis marker dextran and not with the clathrin pathway marker transferrin or with caveolin. Dynasore, an inhibitor of dynamin, did not block viral entry into endothelial cells but did inhibit entry into HFF cells. KSHV was not associated with the early endosome marker EEA-1 in HMVEC-d cells, but rather with the late endosome marker LAMP1, as well as with Rab34 GTPase that is known to regulate macropinocytosis. Silencing Rab34 with small interfering RNA dramatically inhibited KSHV gene expression. Bafilomycin-mediated disruption of endosomal acidification inhibited viral gene expression. Taken together, these findings suggest that KSHV utilizes the actin polymerization-dependent, dynamin-independent macropinocytic pathway that involves a Rab34 GTPase-dependent late endosome and low-pH environment for its infectious entry into HMVEC-d and HUVEC cells. These studies also demonstrate that KSHV utilizes different modes of endocytic entry in fibroblast and endothelial cells.

Efficient gene transfer by pullulan-spermine occurs through both clathrin- and raft/caveolae-dependent mechanisms.

The feasibility and mechanism of gene delivery by pullulan-spermine, a recently developed cationic polysaccharide, were investigated. Pullulan-spermine-mediated transfection of plasmid DNA resulted in greatly reduced cytotoxicity and a 10-fold increase in the level of gene expression when compared to Lipofectamine 2000, a commercially available cationic lipid. Additionally, after transfection of p53-expressing plasmid DNA by pullulan-spermine but not Lipofectamine 2000, the in vitro proliferation of T24 cells was significantly reduced. Pullulan-spermine-mediated gene expression was inhibited by both chlorpromazine of clathrin-mediated endocytosis inhibitor and methyl-beta-cyclodextrin and filipin of raft/caveolae inhibitors. We conclude that pullulan-spermine is a promising carrier for gene transfection, and that cellular uptake of pullulan-spermine-plasmid DNA complexes is mediated by clathrin- and raft/caveolae-dependent endocytotic pathways.

The effect of enamel matrix derivative on gene expression in osteoblasts.

Observations that amelogenins, in the form of enamel matrix derivative (EMD), have a stimulatory effect on mesenchymal cells and tissues, and on the regeneration of alveolar bone, justified investigations into the effect of EMD on bone-forming cells. The binding and uptake of EMD in primary osteoblastic cells was characterized, and the effect of EMD on osteoblast gene expression, protein secretion, and mineralization was compared with the effect of parathyroid hormone (PTH). Although no specific receptor(s) has yet been identified, EMD appeared to be taken up by osteoblasts through clathrin-coated pits via the interaction with clathrin adaptor protein complex AP-2, the major mechanism of cargo sorting into coated pits in mammalian cells. EMD had a positive effect on factors involved in mineralization in vitro, causing an increased alkaline phosphatase (ALP) activity in the medium as well an as increased expression of osteocalcin and collagen type 1. Several hundred genes are regulated by EMD in primary human osteoblasts. There appear to be similarities between the effects of EMD and PTH on human osteoblasts. The expression pattern of several mRNAs and proteins upon EMD stimulation also indicates a secondary osteoclast stimulatory effect, suggesting that the osteogenic effect of EMD in vivo, at least partly, involves stimulation of bone remodelling.

Targeted chemical disruption of clathrin function in living cells.

The accurate assignment of molecular roles in membrane traffic is frequently complicated by the lack of specific inhibitors that can work on rapid time scales. Such inhibition schemes would potentially avoid the complications arising from either compensatory gene expression or the complex downstream consequences of inhibition of an important protein over long periods (>12 h). Here, we developed a novel chemical tool to disrupt clathrin function in living cells. We engineered a cross-linkable form of clathrin by using an FK506-binding protein 12 (FKBP)-clathrin fusion protein that is specifically oligomerized upon addition of the cell-permeant cross-linker FK1012-A. This approach interrupts the normal assembly-disassembly cycle of clathrin lattices and results in a specific, rapid, and reversible approximately 70% inhibition of clathrin function. This approach should be applicable to a number of proteins that must go through an assembly-disassembly cycle for normal function.

A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death.

The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

Calpain inhibitor inhibits secretory granule maturation and secretion of GH.

Clathrin- and AP-1-coated buds are present on immature secretory granules of endocrine cells that mature into clathrin-uncoated granules. The mechanism of clathrin and adaptor protein uncoating has remained obscure. Benzyloxycarbonyl-L-leucyl-L-leucinal (ZLLal), a calpain inhibitor, reduced growth hormone (GH) secretion with intracellular accumulation, in a GH-secreting rat pituitary tumor cell. Pulse and chase demonstrated that ZLLal retarded the turnover of clathrin (Clt.H) and adaptins. ZLLal-treatment co-immunoprecipitated the increased amounts of GH with Clt.H and adaptins compared to control cells, suggesting the intracellular accumulation of immature secretory granules. Clt.H and adaptins were limited-proteolyzed by m-calpain in vitro, indicating that calpain may be involved partly in the maturation of secretory granules in endocrine cells via the process of clathrin uncoating.

Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles.

The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-beta-cyclodextrin (MbetaCD) to selectively extract cholesterol from the plasma membrane. MbetaCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MbetaCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MbetaCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MbetaCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.

Insulin-sensitive association of GLUT-4 with endocytic clathrin-coated vesicles revealed with the use of brefeldin A.

The interaction of the adipocyte/skeletal muscle glucose transporter (GLUT-4) with clathrin lattices may be important in maintaining its intracellular distribution. To better understand the role of clathrin lattices in the sorting of GLUT-4, we have attempted to determine the cellular origin of clathrin-coated vesicles (CCVs) that contain this transporter. The fungal toxin brefeldin A (BFA) causes the selective disassembly of clathrin lattices at the trans-Golgi network (TGN), but not at the plasma membrane (PM), thus providing a way of estimating the proportion of GLUT-4 in PM- versus TGN-derived clathrin lattices. Exposure of 3T3-L1 adipocytes to BFA resulted in a rapid disassembly of clathrin lattices at the TGN, observed by optical sectioning microscopy, and to a pronounced decrease in the yield of CCVs purified from these cells. Thus, CCVs isolated from BFA-treated cells are likely to be derived from the PM. Immunoblotting experiments revealed the presence of GLUT-4 in such CCVs, suggesting that under basal conditions the transporter is continually retrieved from the PM through the CCV pathway. Exposure of both BFA-treated or non-treated cells to insulin resulted in a 4-6-fold increase in the concentration of GLUT-4 at the PM. In parallel, the concentration of GLUT-4 in PM-derived CCVs decreased by 60%. These results suggest (a) that the effect of insulin to increase the cell surface concentration of GLUT-4 is not inhibited by BFA, and (b) that a decreased association of GLUT-4 with endocytic CCVs may be important in facilitating its increased cell surface concentration in response to the hormone.