Cryo-EM structure of an essential Plasmodium vivax invasion complex.

Plasmodium vivax is the most widely distributed malaria parasite that infects humans1. P. vivax invades reticulocytes exclusively, and successful entry depends on specific interactions between the P. vivax reticulocyte-binding protein 2b (PvRBP2b) and transferrin receptor 1 (TfR1)2. TfR1-deficient erythroid cells are refractory to invasion by P. vivax, and anti-PvRBP2b monoclonal antibodies inhibit reticulocyte binding and block P. vivax invasion in field isolates2. Here we report a high-resolution cryo-electron microscopy structure of a ternary complex of PvRBP2b bound to human TfR1 and transferrin, at 3.7 Å resolution. Mutational analyses show that PvRBP2b residues involved in complex formation are conserved; this suggests that antigens could be designed that act across P. vivax strains. Functional analyses of TfR1 highlight how P. vivax hijacks TfR1, an essential housekeeping protein, by binding to sites that govern host specificity, without affecting its cellular function of transporting iron. Crystal and solution structures of PvRBP2b in complex with antibody fragments characterize the inhibitory epitopes. Our results establish a structural framework for understanding how P. vivax reticulocyte-binding protein engages its receptor and the molecular mechanism of inhibitory monoclonal antibodies, providing important information for the design of novel vaccine candidates.

Molecular and nanoscale evaluation of N-cadherin expression in invasive bladder cancer cells under control conditions or GW501516 exposure.

N-cadherin is a transmembrane glycoprotein expressed by mesenchymal origin cells and is located at the adherens junctions. It regulates also cell motility and contributes to cell signaling. In previous studies, we identified that its anomalous expression in bladder carcinoma was a tumor progression marker. A pharmacological approach to inhibit N-cadherin expression or to block its function could be relevant to prevent disease progression and metastasis development. The morphological exploration of T24 invasive bladder cancer cells by atomic force microscopy (AFM) revealed a spindle-like shape with fibrous structures. By engaging force spectroscopy with AFM tip functionalized with anti-E or anti-N-cadherin antibodies, results showed that T24 cells expressed only N-cadherin as also demonstrated by Western blotting and confocal microscopy. For the first time, we demonstrated by RTqPCR and Western blotting analyses that the peroxisome proliferator-activated receptor ß/δ (PPARß/δ) agonist GW501516 significantly decreased N-cadherin expression in T24 cells. Moreover, high non-cytotoxic doses of GW501516 inhibited confluent T24 cell wound healing closure. By using AFM, a more sensitive nanoanalytical method, we showed that the treatment modified the cellular morphology and diminished N-cadherin cell surface coverage through the decreasing of these adhesion molecule-mediated interaction forces. We observed a greater decrease of N-cadherin upon GW501516 exposure with AFM than that detected with molecular biology techniques. AFM was a complementary tool to biochemical techniques to perform measurements on living cells at the nanometer resolution level. Taken together, our data suggest that GW501516 could be an interesting therapeutic strategy to avoid bladder cancer cell spreading through N-cadherin decrease.

pH-Dependent recognition of apoptotic and necrotic cells by the human dendritic cell receptor DEC205.

Dendritic cells play important roles in regulating innate and adaptive immune responses. DEC205 (CD205) is one of the major endocytotic receptors on dendritic cells and has been widely used for vaccine generation against viruses and tumors. However, little is known about its structure and functional mechanism. Here we determine the structure of the human DEC205 ectodomain by cryoelectron microscopy. The structure shows that the 12 extracellular domains form a compact double ring-shaped conformation at acidic pH and become extended at basic pH. Biochemical data indicate that the pH-dependent conformational change of DEC205 is correlated with ligand binding and release. DEC205 only binds to apoptotic and necrotic cells at acidic pH, whereas live cells cannot be recognized by DEC205 at either acidic or basic conditions. These results suggest that DEC205 is an immune receptor that recognizes apoptotic and necrotic cells specifically through a pH-dependent mechanism.

Immunoelectron microscopic evidence for Tetherin/BST2 as the physical bridge between HIV-1 virions and the plasma membrane.

Tetherin/BST2 was identified in 2008 as the cellular factor responsible for restricting HIV-1 replication at a very late stage in the lifecycle. Tetherin acts to retain virion particles on the plasma membrane after budding has been completed. Infected cells that express large amounts of tetherin display large strings of HIV virions that remain attached to the plasma membrane. Vpu is an HIV-1 accessory protein that specifically counteracts the restriction to virus release contributed by tetherin. Tetherin is an unusual Type II transmembrane protein that contains a GPI anchor at its C-terminus and is found in lipid rafts. The leading model for the mechanism of action of tetherin is that it functions as a direct physical tether bridging virions and the plasma membrane. However, evidence that tetherin functions as a physical tether has thus far been indirect. Here we demonstrate by biochemical and immunoelectron microscopic methods that endogenous tetherin is present on the viral particle and forms a bridge between virion particles and the plasma membrane. Endogenous tetherin was found on HIV particles that were released by partial proteolytic digestion. Immunoelectron microscopy performed on HIV-infected T cells demonstrated that tetherin forms an apparent physical link between virions and connects patches of virions to the plasma membrane. Linear filamentous strands that were highly enriched in tetherin bridged the space between some virions. We conclude that tetherin is the physical tether linking HIV-1 virions and the plasma membrane. The presence of filaments with which multiple molecules of tetherin interact in connecting virion particles is strongly suggested by the morphologic evidence.

Simian immunodeficiency virus envelope glycoprotein counteracts tetherin/BST-2/CD317 by intracellular sequestration.

Tetherin is an IFN-inducible restriction factor that inhibits HIV-1 particle release in the absence of the HIV-1 countermeasure, viral protein U (Vpu). Although ubiquitous in HIV-1 and simian immunodeficiency viruses from chimpanzees, greater spot nosed monkeys, mustached monkeys, and Mona monkeys, other primate lentiviruses do not encode a Vpu protein. Here we demonstrate that SIV from Tantalus monkeys (SIVtan) encodes an envelope glycoprotein (SIVtan Env) able to counteract tetherin from Tantalus monkeys, rhesus monkeys, sooty mangabeys, and humans, but not from pigs. We show that sensitivity to Vpu but not SIVtan Env can be transferred with the human tetherin transmembrane region. We also identify a mutation in the tetherin extracellular domain, which almost completely abolishes sensitivity of human tetherin to SIVtan Env without compromising antiviral activity or sensitivity to Vpu. SIVtan Env expression results in a reduction of surface tetherin, as well as reduction in tetherin co-localization with mature surface-associated virus. Immuno-electron microscopy reveals co-localization of SIVtan Env with tetherin in intracellular tubulo-vesicular structures, suggesting that tetherin is sequestered away from budding virions at the cell surface. Along with HIV-1 Vpu and SIV Nef, envelope glycoprotein is the third and most broadly active lentiviral-encoded tetherin countermeasure to be described. Our observations emphasize the importance of tetherin in protecting mammals against viral infection and suggest that HIV-1 Vpu inhibitors may select active envelope mutants.

CD9 clustering and formation of microvilli zippers between contacting cells regulates virus-induced cell fusion.

Members of the tetraspanin family including CD9 contribute to the structural organization and plasticity of the plasma membrane. K41, a CD9-specific monoclonal antibody, inhibits the release of HIV-1 and canine distemper virus (CDV)- but not measles virus (MV)-induced cell-cell fusion. We now report that K41, which recognizes a conformational epitope on the large extracellular loop of CD9, induces rapid relocation and clustering of CD9 in net-like structures at cell-cell contact areas. High-resolution analyses revealed that CD9 clustering is accompanied by the formation of microvilli that protrude from either side of adjacent cell surfaces, thus forming structures like microvilli zippers. While the cellular CD9-associated proteins beta(1)-integrin and EWI-F were co-clustered with CD9 at cell-cell interfaces, viral proteins in infected cells were differentially affected. MV envelope proteins were detected within CD9 clusters, whereas CDV proteins were excluded from CD9 clusters. Thus, the tetraspanin CD9 can regulate cell-cell fusion by controlling the access of the fusion machinery to cell contact areas.

Physiologic relevance of the membrane attack complex inhibitory protein CD59 in human seminal plasma: CD59 is present on extracellular organelles (prostasomes), binds cell membranes, and inhibits complement-mediated lysis.

We demonstrate here that CD59, an inhibitor of the membrane attack complex (MAC) of the complement system, is present in cell-free seminal plasma (SP) at a concentration of at least 20 micrograms/ml. Analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and Edman degradation indicated that this protein, SP CD59, was similar, if not identical, to CD59 isolated from erythrocyte (E) membranes (E CD59). Like purified E CD59, SP CD59 also possesses a glycosyl phosphatidyl inositol (GPI) anchor and incorporates into the membranes of heterologous cells where it inhibits lysis by the human MAC. This phenomenon could be demonstrated not only if cells were incubated with purified SP CD59 but also if unfractionated SP were used. Further, CD59 in unfractionated SP bound to washed spermatozoa, increasing their membrane content of the protein. The mechanism by which this protein retains its GPI anchor while apparently present in the fluid phase is of interest and was further investigated. Using the techniques of high-speed centrifugation, fast performance liquid chromatography fractionation, and electron microscopy, we found that all detectable SP CD59 was associated with vesicular extracellular organelles. These organelles, named "prostasomes," were previously known to be present in SP and to interact with spermatozoa, although their function was uncertain. Interaction of heterologous E with prostasomes rendered the cells more resistant to lysis by human MACs. We propose that these organelles represent a pool of CD59 from which protein lost from spermatozoa, perhaps as a result of low level complement attack or of normal membrane turnover, can be replenished.

Tetraspanin family protein CD9 in the mouse sperm: unique localization, appearance, behavior and fate during fertilization.

A tetraspanin family protein, CD9, has not previously been identified in sperm cells. Here, we characterize sperm CD9 in the mouse, including its unique localization in sperm, appearance during spermatogenesis, and behavior and fate during mouse fertilization. In sperm, CD9 is an inner acrosomal membrane-associated protein, not a plasma membrane-associated protein. Its molecular weight is approximately 24 kDa throughout its processing, from testicular germ cells to acrosome-reacted sperm. A temporal difference was found between mRNA and protein expression; CD9 mRNA was detected in the stages from spermatogonia through round spermatids showing the strongest levels in midpachytene spermatocytes. CD9 protein was detected in the cytoplasm throughout the stages from spermatogonia to spermatocytes. While CD9 was weakly expressed in the spermatids from step 1 through step 14, the signals became clearly positive at the marginal region of the anterior acrosome in elongated spermatids. After the acrosome reaction, the majority of sperm CD9 was retained in the inner acrosomal membrane, but some quantity of CD9 was found on the plasma membrane covering the equatorial segment as detected by immunogold electron microscopy using anti-CD9 antibody. CD9 was maintained on the sperm head after reaching the perivitelline space of CD9-deficient eggs that were recovered after natural mating with wild males. Thus, this study characterizes CD9 in sperm development and fertilization.

Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F.

Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into tetraspanin-enriched microdomains is unknown. Here, we present crystal structures of the large extracellular loop of CD9 bound to nanobodies 4C8 and 4E8 and, the cryo-EM structure of 4C8-bound CD9 in complex with its partner EWI-F. CD9-EWI-F displays a tetrameric arrangement with two central EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at loops C and D, which is in agreement with the 4C8 conformation in the CD9-EWI-F complex. The complex varies from nearly twofold symmetric (with the two CD9 copies nearly anti-parallel) to ca. 50° bent arrangements. This flexible arrangement of CD9-EWI-F with potential CD9 homo-dimerization at either end provides a "concatenation model" for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.

Structural studies of langerin and Birbeck granule: a macromolecular organization model.

Dendritic cells, a sentinel immunity cell lineage, include different cell subsets that express various C-type lectins. For example, epidermal Langerhans cells express langerin, and some dermal dendritic cells express DC-SIGN. Langerin is a crucial component of Birbeck granules, the Langerhans cell hallmark organelle, and may have a preventive role toward HIV, by its internalization into Birbeck granules. Since langerin carbohydrate recognition domain (CRD) is crucial for HIV interaction and Birbeck granule formation, we produced the CRD of human langerin and solved its structure at 1.5 A resolution. On this basis gp120 high-mannose oligosaccharide binding has been evaluated by molecular modeling. Hydrodynamic studies reveal a very elongated shape of recombinant langerin extracellular domain (ECD). A molecular model of the langerin ECD, integrating the CRD structure, has been generated and validated by comparison with hydrodynamic parameters. In parallel, Langerhans cells were isolated from human skin. From their analysis by electron microscopy and the langerin ECD model, an ultrastructural organization is proposed for Birbeck granules. To delineate the role of the different langerin domains in Birbeck granule formation, we generated truncated and mutated langerin constructs. After transfection into a fibroblastic cell line, we highlighted, in accordance with our model, the role of the CRD in the membrane zipping occurring in BG formation as well as some contribution of the cytoplasmic domain. Finally, we have shown that langerin ECD triggering with a specific mAb promotes global rearrangements of LC morphology. Our results open the way to the definition of a new membrane deformation mechanism.

Architecture of the VE-cadherin hexamer.

Vascular endothelial-cadherin (VE-cadherin) is the major constituent of the adherens junctions of endothelial cells and plays a key role in angiogenesis and vascular permeability. The ectodomains EC1-4 of VE-cadherin are known to form hexamers in solution. To examine the mechanism of homotypic association of VE-cadherin, we have made a 3D reconstruction of the EC1-4 hexamer using electron microscopy and produced a homology model based on the known structure of C-cadherin EC1-5. The hexamer consists of a trimer of dimers with each N-terminal EC1 module making an antiparallel dimeric contact, and the EC4 modules forming extensive trimeric interactions. Each EC1-4 molecule makes a helical curve allowing some torsional flexibility to the edifice. While there is no direct evidence for the existence of hexamers of cadherin at adherens junctions, the model that we have produced provides indirect evidence since it can be used to explain some of the disparate results for adherens junctions. It is in accord with the X-ray and electron microscopy results, which demonstrate that the EC1 dimer is central to homotypic cadherin interaction. It provides an explanation for the force measurements of the interaction between opposing cadherin layers, which have previously been interpreted as resulting from three different interdigitating interactions. It is in accord with observations of native junctions by cryo-electron microscopy. The fact that this hexameric model of VE-cadherin can be used to explain more of the existing data on adherens junctions than any other model alone argues in favour of the existence of the hexamer at the adherens junction. In the context of the cell-cell junction these cis-trimers close to the membrane, and trans-dimers from opposing membranes, would increase the avidity of the bond.

Structural model of human endoglin, a transmembrane receptor responsible for hereditary hemorrhagic telangiectasia.

Endoglin is a type I membrane protein expressed as a disulphide-linked homodimer on human vascular endothelial cells whose haploinsufficiency is responsible for the dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia (HHT). Structurally, endoglin belongs to the zona pellucida (ZP) family of proteins that share a ZP domain of approximately 260 amino acid residues at their extracellular region. Endoglin is a component of the TGF-beta receptor complex, interacts with the TGF-beta signalling receptors types I and II, and modulates cellular responses to TGF-beta. Here, we have determined for the first time the three-dimensional structure of the approximately 140 kDa extracellular domain of endoglin at 25 A resolution, using single-particle electron microscopy (EM). This reconstruction provides the general architecture of endoglin, which arranges as a dome made of antiparallel oriented monomers enclosing a cavity at one end. A high-resolution structure of endoglin has also been modelled de novo and found to be consistent with the experimental reconstruction. Each subunit comprises three well-defined domains, two of them corresponding to ZP regions, organised into an open U-shaped monomer. This domain arrangement was found to closely resemble the overall structure derived experimentally and the three modelled de novo domains were tentatively assigned to the domains observed in the EM reconstruction. This molecular model was further tested by tagging endoglin's C terminus with an IgG Fc fragment visible after 3D reconstruction of the labelled protein. Combined, these data provide the structural framework to interpret endoglin's functional domains and mutations found in HHT patients.

Potential mechanism for recruitment and migration of CD133 positive cells to areas of vascular inflammation.

OBJECTIVE: Mast cells are found in large numbers in atherosclerotic plaques. The present study was conducted to determine whether tryptase stimulation of human coronary artery endothelial cells (HCAEC) would lead to an increase in transmigration of CD133 positive cells (CD133+). In vitro these cells can differentiate into mast cells under the influence of specific cytokines and growth factors. METHODS AND RESULTS: CD133+ cells were isolated from umbilical cord blood. They express mRNA for several adhesion molecules that are also utilized in neutrophil migration and can migrate across an HCAEC monolayer. Migration increased significantly when HCAEC were stimulated with tryptase and decreased when CD133+ cells were pretreated with CV3988, a platelet activating factor receptor (PTAFR) antagonist. Following long-term cell culture, these cells stained positively for the presence of tryptase, a mast cell enzyme. CONCLUSION: CD133+ cells can be utilized as a mast cell precursor population. The transendothelial migration is facilitated by the presence of tryptase and may utilize the PAF/PTAFR interaction in a manner similar to that involved in neutrophil transmigration. Following transmigration, a subset of these progenitor cells may mature into mast cells in the subendothelial space and play a role in propagation of the inflammatory process in atherosclerosis.

Visualization of ligand-induced transmembrane signaling in the full-length human insulin receptor.

Insulin receptor (IR) signaling plays a critical role in the regulation of metabolism and growth in multicellular organisms. IRs are unique among receptor tyrosine kinases in that they exist exclusively as covalent (αß)2 homodimers at the cell surface. Transmembrane signaling by the IR can therefore not be based on ligand-induced dimerization as such but must involve structural changes within the existing receptor dimer. In this study, using glycosylated full-length human IR reconstituted into lipid nanodiscs, we show by single-particle electron microscopy that insulin binding to the dimeric receptor converts its ectodomain from an inverted U-shaped conformation to a T-shaped conformation. This structural rearrangement of the ectodomain propagates to the transmembrane domains, which are well separated in the inactive conformation but come close together upon insulin binding, facilitating autophosphorylation of the cytoplasmic kinase domains.

RANKL-induced expression of tetraspanin CD9 in lipid raft membrane microdomain is essential for cell fusion during osteoclastogenesis.

UNLABELLED: We showed that CD9, a member of tetraspanin superfamily proteins, is expressed in a specific membrane microdomain, called "lipid raft," and is crucial for cell fusion during osteoclastogenesis after activation of the RANK/RANKL system. INTRODUCTION: Osteoclasts are bone-resorbing multinuclear polykaryons that are essential for bone remodeling and are formed through cell fusion of mononuclear macrophage/monocyte lineage precursors. Although osteoclastogenesis has been shown to be critically regulated by the RANK/RANKL system, the mechanism how precursor cells fuse with each other remains unclear. We examined the function of CD9, a member of tetraspanin superfamily, which has previously been shown to form macromolecular membrane microdomains and to regulate cell-cell fusion in various cell types. MATERIALS AND METHODS: We used RAW264.7, a macrophage/monocyte lineage cell line, which can differentiate into osteoclast-like polykaryons on the application of RANKL. Expression and distribution of CD9 was assessed by Western blotting, fluorescence-assorted cell sorting (FACS) and immunohistochemistry with light and electron microscopy. A specific neutralizing antibody and RNA interference were used to inhibit the function of CD9, and green fluorescent protein (GFP)-CD9 was exogenously expressed to enhance the effect of CD9. The distribution of CD9 in lipid microdomain was examined by biochemical (sucrose density gradient) isolation and imaging technique. RESULTS: CD9 is expressed on cell surfaces of RAW264.7, which is enhanced by RANKL. Targeted inhibition of CD9 decreases the number of osteoclast-like cells. On the other hand, overexpression of CD9 promotes spontaneous cell fusion even in the absence of RANKL. CD9 is localized in detergent-insoluble "lipid raft" microdomain in RANKL stimulation, and disruption of lipid rafts markedly reduces the formation of osteoclast-like polykaryons. Immunohistochemical studies of bone tissues revealed the expression of CD9 in osteoclasts in vivo. CONCLUSIONS: These data suggest that function of tetraspanin CD9 and its expression in lipid rafts are crucial for cell fusion during osteoclastogenesis.

Solution structure of recombinant human interleukin-6.

Interleukin-6 (IL-6) is a 185 amino acid cytokine which exerts multiple biological effects in vivo and whose dysregulation underlies several disease processes. The solution structure of recombinant human interleukin-6 has now been determined using heteronuclear three and four-dimensional NMR spectroscopy. The structure of the molecule was determined using 3044 distance and torsion restraints derived by NMR spectroscopy to generate an ensemble of 32 structures using a combined distance geometry/simulated annealing protocol. The protein contains five alpha-helices interspersed with variable-length loops; four of these helices constitute a classical four-helix bundle with the fifth helix located in the CD loop. There were no distance violations greater than 0.3 A in any of the final 32 structures and the ensemble has an average-to-the-mean backbone root-mean-square deviation of 0.50 A for the core four-helix bundle. Although the amino-terminal 19 amino acids are disordered in solution, the remainder of the molecule has a well defined structure that shares many features displayed by other long-chain four-helix bundle cytokines. The high-resolution NMR structure of hIL-6 is used to rationalize available mutagenesis data in terms of a heteromeric receptor complex.

High-resolution CryoFESEM of individual cell adhesion molecules (CAMs) in the glycocalyx of human platelets: detection of P-selectin (CD62P), GPI-IX complex (CD42A/CD42B alpha,B beta), and integrin GPIIbIIIa (CD41/CD61) by immunogold labeling and stereo imaging.

The aim of this study was to develop a model for the detection of individual cell adhesion molecules (CAMs) in the glycocalyx of spread human platelets using high-resolution cryo-field emission scanning electron microscopy (cryoFESEM). Three surface glycoprotein CAMs, P-selectin (CD62P), GPIba in the GPI-IX complex (CD42a/CD42b alpha,b beta), and the integrin GPIIbIIIa (CD41/CD61) in the human platelet were selected on the basis of their unique topographic shape. Spread human platelets were indirectly immunolabeled with 10-nm colloidal gold and then cryoimmobilized. After sublimation of water from the cryoimmobilized sample, partially freeze-dried platelets were coated unidirectionally with Pt, stabilized with carbon, and examined in an in-lens cryoFESEM using high-resolution backscattered electron imaging. CAMs were detected by indirect immunogold labeling and the length of each type of CAM was determined using analysis of differences in parallax as measured in the software program Sterecon. Our results demonstrate the efficacy of using high-resolution cryoFESEM to recognize and detect individual CAMs in the glycocalyx. Further advances in production of metal coatings with finer granularity, together with improvements in imaging (tilting and angle of stereo images), may provide better definition of the topography associated with glycosylation and formation of multimeric CAM complexes. (J Histochem Cytochem 49:809-819, 2001)

The mechanism by which proteolysis enhances the ligand-binding activity of guinea pig type II Fc receptor for IgG (FcgammaRIIB).

We have previously shown that the ligand-binding activity of type II Fc receptor for IgG (FcgammaRIIB) on guinea pig peripheral blood polymorphonuclear leukocytes is very low and dramatically increases after treatment of the cells with proteolytic enzymes. In the present study, we analyzed the mechanism of this augmentation. We found that the protease treatment failed to enhance the binding of monomeric IgG to FcgammaRIIB, increased the binding of small immune complexes (IC) prepared under antigen-excess conditions only modestly, but markedly enhanced the binding of large IC prepared under antibody-excess conditions. These results suggest that proteolysis increases the ligand-binding avidity but not the intrinsic affinity of FcgammaRIIB. Confocal laser scanning microscopy revealed that the mobility of FcgammaRIIB on the cell surface was increased after protease treatment. In addition, transfection experiments indicated that the effect of proteolysis on IC binding to CHO cells expressing guinea pig FcgammaRIIB was strongly dependent on the receptor density. Finally, we demonstrated that the transmembrane and cytoplasmic domains of FcgammaRIIB were not involved in the proteolysis-induced augmentation of IC binding. Together our results suggest that the mobility of FcgammaRIIB, which may be restricted due to the association of the ectodomain of the receptor with unknown membrane proteins, is enhanced by proteolysis, allowing the receptors to bind multivalent ligands more readily and hence with higher avidity.

Octreotide differentially modulates human Caco-2 intestinal epithelial cell proliferation and differentiation by decreasing intracellular cAMP.

Somatostatin modulates gastrointestinal mucosal growth and differentiation indirectly via inhibition of bioactive peptides and directly by less well understood mechanisms. We studied the direct effects of the somatostatin analog octreotide on proliferation, brush-border enzyme activity, cell-matrix interactions and intracellular cAMP in Caco-2 human intestinal epithelial cells. Proliferation was assessed by cell counting and [3H]thymidine uptake. The brush-border enzymes alkaline phosphatase (AP) and dipeptidyl dipeptidase (DP) were quantitated by synthetic substrate digestion. Adhesion and migration on purified matrix proteins were also measured. Octreotide (10(-9)-10(-5)M) shortened doubling time (46.5 +/- 6.2% at 10(-5) M, n = 20, P < 0.0001) and stimulated [3H]thymidine uptake. Octreotide decreased intracellular cAMP by 19.4 +/- 5.0% (n = 7, P < 0.0001) while dibutyryl-cAMP (10(-6) M) prolonged doubling time by 10.1 +/- 1.5% (n = 8, P < 0.0001), and blocked the octreotide effect. Octreotide decreased AP and DP with maximal effect at 10(-6) M (36.8 +/- 8.3% and 20.5 +/- 9.1%, n > 7, P < 0.0005 respectively). However, mitomycin proliferative blockade prevented octreotide inhibition of AP and DP, suggesting that the mitogenic effects of octreotide had simply decreased average maturity of the cells. Octreotide did not alter Caco-2 adhesion, EGF-or matrix-modulated motility, or integrin surface expression. Octreotide appears to directly stimulate Caco-2 proliferation by decreasing cAMP. These proliferative effects modulate Caco-2 differentiation but do not affect cell-matrix interactions.