Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2.

Apoptosis is implicated in the generation and resolution of inflammation in response to bacterial pathogens. All bacterial pathogens produce lipoproteins (BLPs), which trigger the innate immune response. BLPs were found to induce apoptosis in THP-1 monocytic cells through human Toll-like receptor-2 (hTLR2). BLPs also initiated apoptosis in an epithelial cell line transfected with hTLR2. In addition, BLPs stimulated nuclear factor-kappaB, a transcriptional activator of multiple host defense genes, and activated the respiratory burst through hTLR2. Thus, hTLR2 is a molecular link between microbial products, apoptosis, and host defense mechanisms.

Effect of parasympathetic denervation on acetylcholine levels in the rat parotid gland. Is there an extraneuronal pool of acetylcholine?

Parasympathetic denervation of the rat parotid gland by avulsion of the auriculotemporal nerve caused a marked and lasting decrease in gland weight. Parasympathectomy did not change the levels of choline in the gland but decreased by 60% the levels of acetylcholine (ACh) ten days after surgery and 65% at 28 days. It is puzzling that relatively high levels of ACh remained after parasympathetic denervation. The presence of additional cholinergic nerves that innervate the gland, or pass through it en route to other structures may account for some of the remaining ACh. Also, Schwann cells from denervated nerves might have contributed to some of the ACh. The existence of an extraneuronal source of ACh is considered.

Effect of clonidine on secretion of fluid and ions by the parotid and submandibular glands of the rat.

Secretion in response to this alpha 2-adrenergic agonist was evaluated in the presence and absence of several adrenergic antagonists, reserpine and sympathectomy (Sx). In both glands, the response was qualitatively but not quantitatively similar to that induced by the alpha 1-adrenergic agonist, phenylephrine, in the presence of propranolol. With clonidine, the volume of submandibular saliva was much higher but the Ca concentration was 3-4 times lower than that of the parotid; both salivas had low Na but high K concentrations. Clonidine-induced secretion was almost completely blocked by the alpha 1-adrenergic antagonist, prazosin and a mixed alpha-adrenergic antagonist, phentolamine, and markedly reduced by the alpha 2-adrenergic antagonist, yohimbine, but unaffected by the beta-adrenergic antagonist, propranolol. Reserpine reduced the parotid, but enhanced the submandibular secretory response to clonidine. Results in Sx glands were similar. Thus, in the rat glands clonidine may activate alpha 1-rather than alpha 2-adrenoceptors, which appear to play a part similar to alpha 1-adrenoceptors only after reserpine or Sx.

Amino acid uptake in the rat parotid gland: II. Effects of surgical sympathectomy and parasympathectomy.

The effects of surgical denervation and pharmacologic stimulation on amino acid transport have been investigated. Uptake of a nonmetabolizable amino acid, [14C]alpha-aminoisobutyric acid (AIB), was measured in an in vitro system of parotid lobules prepared from surgically denervated glands. Despite atrophic changes indicated by morphologic and biochemical examination, lobules prepared from sympathectomized glands showed a 22% (P less than .05) increase in [14C]AIB uptake. Uptake by lobules prepared from parasympathectomized glands had a similar average increase, but results were more variable (P greater than .05). Analysis of initial flux data for AIB suggested that a small increase in Vmax and a small decrease in Km occur after sympathetic or parasympathetic denervation. Norepinephrine, at a concentration of 10(-6) M, caused acceleration of AIB uptake in normal lobules (60%) and further increase in parasympathectomized lobules (150%) and sympathectomized lobules (200%). Acceleration of AIB uptake was only evident, however, after 1 hr of incubation, suggesting an intermediate event. The pattern of in vitro uptake of [3H]norepinephrine over a period of 3 hr was studied and revealed a biphasic pattern of uptake in control and parasympathectomized tissue. Early rapid increase in radioactivity was followed by slow decline. A different pattern was observed in sympathectomized glands, in which radioactivity increased steadily but at a reduced rate. These results suggest that the initial rapid phase of uptake may be the result of neuronal uptake followed by gradual degradation and release of radioactive metabolites. [3H]Norepinephrine uptake by parasympathectomized glands averaged 20% greater than controls, suggesting a possible expansion of the sympathetic component after surgery.

Amino acid uptake in denervated rat parotid gland: I. Preparation and evaluation of a lobule system for in vitro studies.

The preparation and evaluation of an in vitro system to study the effects of denervation on amino acid uptake in the adolescent rat parotid gland are described. The biochemical consequences of denervation in adolescent rats were different from those seen in the adult. Surgical sympathectomy at 23 to 25 days resulted in marked decreases in weight (29%), acid-precipitable protein (21%) and alpha-amylase (50%) 2 weeks after surgery. Measurement of DNA indicated a loss of cells but no change in cell volume. These effects of sympathectomy were reversible, and these parameters had returned to control levels at 4 weeks. Parasympathetic denervation resulted in decreases in weight (48%), protein (20%) and alpha-amylase (30%) 2 weeks after surgery and paralleled those seen in the adult rat. Unlike sympathectomized glands, the deficits were not reversed at 4 weeks. Fluorometric measurement of norepinephrine and in vivo uptake of [3H]norepinephrine showed a large depletion at 2 weeks (80 and 84%, respectively). Auriculotemporal neurotomy (parasympathectomy), assessed by gas chromatographic measurement of acetylcholine, resulted in a 60% decrease in acetylcholine. The evaluation of a lobule preparation of these glands, suitable for short-term in vitro studies, is also described. Lobules were prepared by strictly mechanical means to avoid membrane changes reported in in vitro systems using enzymatically dissociated tissue. Histologic evaluation indicated that most lobules were free of mechanical damage, and normal secretory units were intact.(ABSTRACT TRUNCATED AT 250 WORDS)

Short-term primary culture of acinar-intercalated duct complexes from rat submandibular glands.

Acinar-intercalated duct complexes dissociated from rat submandibular glands have been shown to be an excellent model for studying secretory responses of salivary gland components. However, they are functionally normal for only a few hours. We undertook a systematic manipulation of primary culture conditions in an attempt to extend the useful life of the complexes. The major modifications that were tested were increased oxygenation in increments to 95%; substitution of norepinephrine or carbamylcholine or both for isoproterenol in the medium; different sources of collagen for and addition of laminin, fibronectin and/or type IV collagen to the matrix gel; and varying the thickness of the collagen gel, richness of the cell suspension inoculate, and sources and concentrations of sera in the medium. Progress was monitored by light microscopic evaluation of routine sections of specimens until improved maintenance of acinar and other cells warranted carrying parallel cultures for biochemical, histochemical, and ultrastructural analyses. Best results were obtained with 90% O2, laminin in rat tail collagen gel, 10% fetal bovine serum, and 3 microM isoproterenol. Morphologically, there was good survival of acini and intercalated ducts after 1 d, with decreased acinar size being correlated with secretory response to the isoproterenol. Reorganization and considerable mitotic activity were seen at 2, 3, and 4 d, with most clusters of cells becoming much larger than the original complexes. During this period acinar cells steadily became less differentiated and their numbers decreased in proportion to intercalated duct or undifferentiated cells. However, there was good overall survival through 7 d. Biochemical analysis indicated that the cells were able to maintain significant biosynthetic activity for 4 d, with DNA, RNA, protein, and glycoprotein synthetic rates increasing over the culture period, but the secretory capacity of the cells diminished during the primary culture period, with mucin biosynthesis and secretion decreasing significantly after 1 d in culture.

Characterization of Gas6, a member of the superfamily of G domain-containing proteins, as a ligand for Rse and Axl.

Rse, Ax1, and c-Mer comprise a family of cell adhesion molecule-related tyrosine kinase receptors. Human Gas6 was recently shown to act as a ligand for both human Rse (Godowski et al., 1995) and human Ax1 (Varnum et al., 1995). Gas6 contains an NH2-terminal Gla domain followed by four epidermal growth factor-like repeats and tandem globular (G) domains. The G domains are related to those found in sex hormone-binding globulin and to those utilized by laminin and agrin for binding to the dystroglycan complex. A series of Gas6 variants were tested for their ability to bind to Rse and Ax1. The Gla domain and epidermal growth factor-like repeats were not required for receptor binding, as deletion variants of Gas6 which lacked these domains bound to the extracellular domains of both Rse and Axl. A deletion variant of Gas6 containing just the G domain region was shown to activate Rse phosphorylation. These results provide evidence that G domains can act as signaling molecules by activating transmembrane receptor tyrosine kinases. Furthermore, they provide a structural link between the activation of cell adhesion related receptors and the control of cell growth and differentiation by the G domain-containing superfamily of proteins.

Ecdysteroid-dependent regulation of genes in mammalian cells by a Drosophila ecdysone receptor and chimeric transactivators.

Steroid receptors are members of a large family of transcription factors whose activity is tightly regulated by the binding of their cognate steroid ligand. Mammalian steroid hormone receptors have been exploited to obtain the regulated expression of heterologous genes in mammalian cells. However, the utility of these systems in cultured cells and transgenic animals is limited by the presence of endogenous steroids and their receptors. We show that a Drosophila ecdysone receptor can function in cultured mammalian cells as an ecdysteroid-dependent transcription factor. The activity of the ecdysone receptor was not induced by any of the mammalian steroid hormones tested. The DNA-binding and transactivation activities of viral, mammalian, or bacterial proteins were rendered ecdysteroid-dependent when fused to the ligand-binding domain of the ecdysone receptor. The ecdysone receptor may prove useful in selectively regulating the expression of endogenous or heterologous genes in mammalian cells.

Signaling events induced by lipopolysaccharide-activated toll-like receptor 2.

Human Toll-like receptor 2 (TLR2) is a signaling receptor that responds to LPS and activates NF-kappaB. Here, we investigate further the events triggered by TLR2 in response to LPS. We show that TLR2 associates with the high-affinity LPS binding protein membrane CD14 to serve as an LPS receptor complex, and that LPS treatment enhances the oligomerization of TLR2. Concomitant with receptor oligomerization, the IL-1R-associated kinase (IRAK) is recruited to the TLR2 complex. Intracellular deletion variants of TLR2 lacking C-terminal 13 or 141 aa fail to recruit IRAK, which is consistent with the inability of these mutants to transmit LPS cellular signaling. Moreover, both deletion mutants could still form complexes with wild-type TLR2 and act in a dominant-negative (DN) fashion to block TLR2-mediated signal transduction. DN constructs of myeloid differentiation protein, IRAK, TNF receptor-associated factor 6, and NF-kappaB-inducing kinase, when coexpressed with TLR2, abrogate TLR2-mediated NF-kappaB activation. These results reveal a conserved signaling pathway for TLR2 and IL-1Rs and suggest a molecular mechanism for the inhibition of TLR2 by DN variants.

The interaction of urea with the generic class of poly(2-hydroxyethyl methacrylate) hydrogels.

Work reported here shows that, contrary to reports in the literature, hydrogels made from pure poly(2-hydroxyethyl methacrylate), pHEMA, at crosslinker content greater than 0.15 mol % do not swell above the usual equilibrium values of 39-42% water content in aqueous urea solution. However, hydrogels containing small (impurity) amounts of methacrylic acid (MAA) do swell dramatically (approximately 90%) in dilute urea solution, but not directly due to the urea. The urea decomposes to produce ammonium ions, thus raising the pH of the solution. Ionization of MAA occurs above pH 6, causing electrostatic interactions within the gel. The grossly swollen state of these gels represents an internal equilibrium among forces due to rubber elasticity, polymer-polymer/solvent affinity, and electrostatic interactions.

Expression and characterization of hepatocyte growth factor receptor-IgG fusion proteins. Effects of mutations in the potential proteolytic cleavage site on processing and ligand binding.

The receptor for hepatocyte growth factor (HGF) is the product of the c-met proto-oncogene, a membrane-spanning tyrosine kinase receptor. To facilitate analysis of HGF and its receptor (HGFr), we expressed and purified a chimeric protein containing the extracellular domain (ECD) of the HGFr fused to the constant region of IgG heavy chain. This soluble form of the HGFr (sHGFr) bound HGF with an affinity similar to that of the authentic, membrane-associated receptor. The sHGFr also neutralized the binding of HGF to the HGFr expressed on A549 cells. Like the mature form of the HGFr, sHGFr is a heterodimer which arises by proteolytic processing within the ECD. In order to characterize the requirements for proteolytic processing of the ECD and the effects of cleavage on ligand binding, we expressed sHGFr variants containing amino acid substitutions in the putative processing site. Replacement of the P1 or P4 arginine, but not the P3 lysine, with alanine inhibited conversion to the alpha/beta heterodimer. This suggests that maturation is mediated by furin or a furin-like protease. Finally, we showed that processing of the sHGFr into the alpha/beta form is not required for high affinity binding to either pro- or mature HGF.

rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain.

We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.

Identification and characterization of SPRK, a novel src-homology 3 domain-containing proline-rich kinase with serine/threonine kinase activity.

Protein kinase play important roles in the growth and differentiation of cells. We have isolated cDNA clones from the human megakaryocytic cell line CMK11-5 that encode a novel protein kinase, which we call SPRK (src-homology 3 (SH3) domain-containing proline-rich kinase). The gene sequence predicts an 847-amino acid protein kinase with a unique domain arrangement. An amino-terminal glycine-rich region is followed by an SH3 domain and a kinase domain that is similar to both tyrosine and serine/threonine kinases. Adjacent to the kinase domain are two closely spaced leucine/isoleucine zipper motifs and a stretch of basic amino acids that resembles karyophilic nuclear localization signals. The COOH-terminal half of SPRK is basic, and proline accounts for 24% of the COOH-terminal 216 amino acids. The sprk gene is widely expressed as a 4-kilobase transcript in adult and fetal human tissues. Transfection of 293 cells with a vector encoding an epitope-tagged SPRK results in the expression of a 95-kDa protein. The epitope-tagged SPRK becomes phosphorylated on serine and threonine residues in an in vitro kinase assay, whereas SPRK variants with point mutations in the predicted ATP-binding site fail to become phosphorylated. These data indicate that SPRK has serine/threonine kinase activity. The SH3 domain of SPRK is interrupted by a unique 5-amino acid insert whose location in the SH3 consensus sequence is the same as that of the inserts found in the SH3 domains of neuronal SRC and of the p85 subunit of phosphatidylinositol 3-kinase.

Structure-function analysis of hepatocyte growth factor: identification of variants that lack mitogenic activity yet retain high affinity receptor binding.

Hepatocyte growth factor (HGF) is a potent mitogen for parenchymal liver, epithelial and endothelial cells. Structurally, it has similarities to kringle-containing serine proteases, although it does not possess proteolytic activity. A structure-activity relationship study of human HGF was performed by functional analysis of HGF substitution and deletion variants. Analysis of HGF variants was accomplished by defining their ability to induce DNA synthesis on hepatocytes in primary culture and to compete with wild-type HGF for binding to a soluble form of the HGF receptor. Three groups of variants were made: (i) substitutions at the cleavage site, (ii) substitutions within the protease-like domain and (iii) deletions of the beta-chain and/or kringle domains. Our results show that: (i) single-chain HGF is a zymogen-like promitogen in that cleavage into a two-chain form is required for biological activity, however, the single chain form of HGF still retains substantial receptor binding capacity; (ii) certain mutations in the protease-like domain result in variants that are completely defective for mitogenic activity, yet exhibit apparent receptor binding affinities similar to wild-type HGF (Kd approximately 50-70 pM); and (iii) a variant containing the N-terminal 272 residues of mature HGF showed only a 4-fold increase in Kd when compared with wild-type HGF indicating that a primary receptor binding determinant is located within this sequence.

Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling.

Vertebrates and invertebrates initiate a series of defence mechanisms following infection by Gram-negative bacteria by sensing the presence of lipopolysaccharide (LPS), a major component of the cell wall of the invading pathogen. In humans, monocytes and macrophages respond to LPS by inducing the expression of cytokines, cell-adhesion proteins, and enzymes involved in the production of small proinflammatory mediators. Under pathophysiological conditions, LPS exposure can lead to an often fatal syndrome known as septic shock. Sensitive responses of myeloid cells to LPS require a plasma protein called LPS-binding protein and the glycosylphosphatidylinositol-anchored membrane protein CD14. However, the mechanism by which the LPS signal is transduced across the plasma membrane remains unknown. Here we show that Toll-like receptor 2 (TLR2) is a signalling receptor that is activated by LPS in a response that depends on LPS-binding protein and is enhanced by CD14. A region in the intracellular domain of TLR2 with homology to a portion of the interleukin (IL)-1 receptor that is implicated in the activation of the IL-1-receptor-associated kinase is required for this response. Our results indicate that TLR2 is a direct mediator of signalling by LPS.