TGF-beta1 up-regulates paxillin protein expression in malignant astrocytoma cells: requirement for a fibronectin substrate.

Cytokines can influence the interactions between members of the integrin cell adhesion receptor family and the extracellular matrix thereby potentially affecting cell function and promoting cell adhesion, growth and migration of malignant astrocytoma tumor cells. As malignant astrocytoma cells synthesize TGF-beta1 in vivo, we analysed the effects of TGF-beta1 on signaling events associated with integrin receptor ligation, focusing on the effects on paxillin, a phosphorylated adaptor protein, that acts as a scaffold for signaling molecules recruited to focal adhesions. TGF-beta1-stimulation of primary astrocytes and serum-starved U-251MG malignant astrocytoma cells attached to fibronectin induced a substantial increase in the levels of paxillin protein (fivefold increase at 2.0 ng/ml) in a dose- and time-dependent manner compared to the levels observed on plating onto fibronectin in the absence of stimulation. In the astrocytoma cells, this resulted in an increase in the pool of tyrosine-phosphorylated paxillin, although it did not appear to alter the extent of phosphorylation of the paxillin molecules. In contrast, in primary astrocytes the protein levels were upregulated in the absence of a parallel increase in phosphorylation. The TGF-beta1-stimulated increase in paxillin levels required ligation of the fibronectin receptor, as it was not induced when the cells were plated onto vitronectin, collagen or laminin. The increase in the pool of paxillin on TGF-beta1 stimulation of the fibronectin-plated astrocytoma cells was associated with an increase in translation, but was not associated with an increase in the steady-state levels of paxillin mRNA. Stimulation with TGF-beta1 on a fibronectin substrate increased subsequent attachment and spreading of U-251MG cells onto fibronectin and, to a lesser extent, vitronectin, but not collagen. Our results indicate that physiologic levels of TGF-beta1 stimulate the expression of paxillin protein at the level of translation through a process that requires engagement of the fibronectin receptor, and promotes attachment and spreading of malignant astrocytoma cells on fibronectin.

Chemoreception in hydra: specific binding of glutathione to a membrane fraction.

Specific binding of reduced [35S]glutathione (GSH) was measured using a crude membrane fraction of Hydra vulgaris (attenuata). The specific binding shows both rapid displaceable and nondisplaceable components. Rapid displaceable binding accounted for 20% of the total specific binding. Data from saturation binding experiments indicates half maximal total specific binding occurs at 2 microM GSH which is similar to reported EC50 values from behavioral experiments. Calcium is required for displaceable binding of GSH to the putative receptor. Oxidized glutathione (GSSG), an antagonist of the GSH-activated feeding response, and S-methylglutathione (GSM), an agonist of the feeding response, inhibit the binding of radiolabeled GSH to the putative receptor. Glutamate, a putative competitive antagonist of the GSH-activated feeding response in hydra, does not inhibit the specific binding of radiolabeled GSH to the receptor and must therefore block the feeding response by a mechanism other than competitive inhibition.

Chemoreception in Hydra vulgaris (attenuata): initial characterization of two distinct binding sites for L-glutamic acid.

To elucidate the relationship between L-glutamic acid and the putative chemoreceptor for glutathione, binding of L-[3H]glutamate to a crude membrane fraction from Hydra vulgaris (attenuata) has been characterized. The binding of L-[3H]glutamate was rapid, reversible and saturable. A Scatchard analysis of the specific binding revealed values of 10 microM for the dissociation constant (Kd) and 170 pmol/mg for the maximal capacity of binding sites (Bmax). A maximum of 65% of the specific L-[3H]glutamate binding was inhibited by the chemostimulatory peptide, glutathione. This glutathione-sensitive glutamate binding presumably represents the association of glutamate with a putative chemoreceptor which modulates feeding behavior in hydra. The remaining 35% of the specific L-[3H]glutamate binding may be due to a second class of glutamate binding sites which is insensitive to glutathione. The identification of glutathione-insensitive glutamate binding is the first indication of a putative glutamate receptor, which may mediate an action independent of the glutathione-induced feeding response. The glutathione-insensitive and glutathione-sensitive sites must have similar affinities for glutamate since these sites were indistinguishable by Scatchard analysis. A preliminary characterization of the glutathione-insensitive site, performed in the presence of saturating levels of glutathione, revealed inhibition of glutathione-insensitive glutamate binding by kainate and quisqualate, but not by N-methyl-D-aspartate. A glutathione-insensitive L-[3H]glutamate binding suggests that kainate and alpha-aminoadipate may be selective ligands for the glutathione-insensitive and glutathione-sensitive glutamate binding sites, respectively.

Regulation of mesenchymal stem cell attachment and spreading on hydroxyapatite by RGD peptides and adsorbed serum proteins.

The successful development of biomaterials must take into consideration how those surfaces will interact with in vivo processes such as adsorption of endogenous proteins. In this study, we examined whether modifying highly adsorbent materials like hydroxyapatite (HA) with RGD peptides would improve mesenchymal stem cell (MSC) adhesion. We found that RGD, alone, was not sufficient to promote full cell spreading. However, given that RGD-modified HA will likely adsorb osteogenic serum proteins in vivo, we evaluated MSC behavior on HA pre-coated with RGD, then over-coated with serum (RGD/FBS). Interestingly, RGD/FBS coatings additively stimulated MSC attachment and spreading compared to either coating alone, but only at low RGD coating concentrations. High RGD concentrations inhibited cell attachment, and completely eliminated cell spreading on RGD/FBS surfaces. To better understand the mechanism by which RGD and adsorbed serum proteins interactively regulate cell behavior, we monitored the deposition of fibronectin (FN) from serum onto HA pre-coated with increasing RGD concentrations. These studies showed that high RGD concentrations did not inhibit FN adsorption, therefore cell spreading is attenuated by mechanisms other than lack of FN availability. Collectively, our results suggest a potential therapeutic benefit for functionalizing HA with RGD, however such a benefit will likely depend upon the RGD density.

The effect of the addition of a polyglutamate motif to RGD on peptide tethering to hydroxyapatite and the promotion of mesenchymal stem cell adhesion.

Mimicking endogenous bone-binding proteins, RGD peptides have been synthesized with polyacidic amino acid domains in order to ionically tether the peptides to bone-like synthetic biomaterials, including hydroxyapatite (HA). However, a direct comparison of unmodified RGD with polyacidic-conjugated RGD has not been performed, and thus a benefit for the acidic domain has not been established. We evaluated the peptide/HA bond of RGD peptides with and without an attached polyglutamate sequence (E(7)), as well as examined mesenchymal stem cell (MSC) adhesion and morphology as they were affected by the conjugated peptide. We found that significantly more E(7)RGD was bound to HA than RGD at all coating concentrations tested, and moreover, more E(7)RGD was retained on the HA surface even after extended washing in serum-free media. Consistent with in vitro results, higher levels of E(7)RGD than RGD remained on HA that had been implanted in vivo for 24 h, indicating that the polyacidic domain improved peptide-binding efficiency. At several peptide concentrations, E(7)RGD increased cell adhesion compared to RGD surfaces, establishing a biological benefit for the E(7) modification. In addition, HA pre-coated sequentially with low-density E(7)RGD (1-10 microg/ml) and serum (FBS) stimulated cell adhesion and spreading, compared to either coating alone, suggesting that an ionic linkage allows for the potential adsorption of serum proteins to unoccupied sites, which may be important for bone formation in vivo. Collectively, these results suggest that tethering peptides to HA via a polyglutamate domain is an effective method for improving the peptide/HA bond, as well as for enhancing MSC adhesion.

Affinity purification of Hydra glutathione binding proteins.

The association of glutathione (GSH) with putative external chemoreceptors elicits feeding behavior in Hydra. In the present study, solubilized membrane proteins were chromatographed on an affinity column of immobilized GSH in order to isolate GSH-binding proteins that may represent the Hydra GSH chemoreceptor. The most abundant of the affinity-purified proteins was a triplet of peptides ranging in molecular weight from 24.5-26 kDa. Antiserum generated against the 24.5-26 kDa triplet peptides inhibited GSH-stimulated feeding behavior by 47%, implicating a role for one or more of these peptides in Hydra chemoreception.

Partial characterization and detergent solubilization of the putative glutathione chemoreceptor from hydra.

Feeding behavior in hydra is initiated by the association of glutathione (GSH) with a putative external chemoreceptor. In the present study, the binding of [35S]GSH to hydra membranes has been characterized. Nondisplaceable [35S]GSH binding which compromised previous analyses [Grosvenor, W., Bellis, S., Kass-Simon, G., & Rhoads, D. (1992) Biochim. Biophys. Acta (in press)] was eliminated by treating membranes with an inhibitor of GSH metabolism, borate in combination with L-serine. The specific binding which was not inhibited by borate/serine demonstrated many of the characteristics expected of a ligand/receptor interaction. The binding was rapid, reversible, and saturable. A Scatchard analysis of saturation isotherms indicated a dissociation constant (KD) of 3.4 microM, a value which is in good agreement with concentrations of glutathione which are known to induce feeding behavior. Hydra membranes were detergent-solubilized with 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 100 mM KCl, and 10% glycerol. The soluble fraction contained 40% of the original saturable, reversible GSH binding activity. The KD for GSH binding to the solubilized preparation was estimated as 2.7 microM, a valuable which is not appreciably different from the KD for binding to intact membranes. The fidelity of GSH binding in the solubilized preparation suggests that this preparation will be useful in further characterization of the putative glutathione chemoreceptor.

Characterization of tyrosine phosphorylation of paxillin in vitro by focal adhesion kinase.

The concomitant tyrosine phosphorylation of the focal adhesion protein, paxillin, and the tyrosine kinase, focal adhesion kinase (FAK), in response to multiple stimuli including integrin-mediated cell adhesion suggests that paxillin phosphorylation is closely coupled to FAK activity. In the present study, we have identified a specific tyrosine residue within paxillin, tyrosine 118 (Tyr-118), that represents the principle site of phosphorylation by FAK in vitro. The identification of this site as a target for FAK phosphorylation was accomplished by immunoprecipitating FAK and performing in vitro kinase assays, using as substrate either glutathione S-transferase (GST)-paxillin fusion proteins containing truncations in paxillin sequence or fusion proteins with phenylalanine substitutions for tyrosine residues. GST-paxillin containing a phenylalanine substitution at Tyr-118 (Y118F) was not phosphorylated by FAK immunoprecipitates; however, this mutant was shown to bind FAK equally as well as the wild type fusion protein. As a first step toward assessing the function of paxillin phosphorylation on Tyr-118, a Y118F paxillin cDNA construct was transiently transfected into NIH 3T3 cells. Similar to wild type paxillin, mutated paxillin localized to focal adhesions, indicating that the phosphorylation of paxillin on Tyr-118 is not essential for the recruitment of paxillin to sites of cell adhesion.

Hydroxylapatite binds more serum proteins, purified integrins, and osteoblast precursor cells than titanium or steel.

The implant material hydroxylapatite (HA) has been shown in numerous studies to be highly biocompatible and to osseointegrate well with existing bone; however, the molecular mechanisms at work behind this osseointegration remain largely unexplored. One possibility is that the implant, exposed to the patient's blood during surgery, adsorbs known cell adhesive proteins such as fibronectin and vitronectin from the serum. Osteoblast precursors could then adhere to these proteins through integrin-mediated mechanisms. In the present study, we have used a quantitative ELISA assay to test the hypothesis that hydroxylapatite will adsorb more fibronectin and vitronectin from serum than two commonly used hard-tissue materials, commercially pure titanium, and 316L stainless steel. We further used the ELISA, as well as a standard cell adhesion assay, to test the hypothesis that increased protein adsorption will lead to better binding of purified integrins alpha5beta1 and alpha(v)beta3 and osteoblast precursor cells to the HA than to the metals. Our results show that fibronectin, vitronectin, alpha5beta1, alpha(v)beta3, and osteoblast precursor cells do indeed bind better to HA than to the metals, suggesting that improved integrin-mediated cell binding may be one of the mechanisms leading to better clinical bone integration with HA-coated implants.

Steps in integrin beta1-chain glycosylation mediated by TGFbeta1 signaling through Ras.

Ras is activated by transforming growth factor beta (TGFbeta) in several cell types, but the biological consequences of this activation are largely unknown. We now show that ras mediates two stages in integrin beta1-chain maturation: 1) glycosylation of the 86-kD core peptide, which is a TGFbeta1-independent process, and 2) TGFbeta1-mediated conversion of the 115-kD beta1 integrin precursor into the mature 130-kD form. HD3 colon epithelial cells maintain elevated levels of integrin alpha2beta1 heterodimers, strong binding to collagen I, and autocrine regulation by TGFbeta1, which converts beta1 integrin into the mature cell surface form. Each of three HD3 cell clones that stably express dominant negative ras (N17ras) exhibited abnormal glycosylation of the integrin beta1-chain, decreased cell surface expression of the mature integrin beta1, and impaired binding to collagen and laminin. Autocrine levels of TGFbeta were not altered by expression of N17ras. The aberrant glycosylation of the integrin beta1-chain was reversed by antisense oligonucleotides specific to the DNA sequence encoding the rasS17N mutation. Glycosylation of the 86-kD core peptide was delayed in the N17ras transfectants, but was not altered by either the addition of TGFbeta1 or inhibition of autocrine TGFbeta1. In contrast, conversion of the partially glycosylated beta1 integrin precursor into the mature 130-kD isoform was accelerated by exogenous TGFbeta1 and blocked by neutralizing antibody to autocrine TGFbeta1 in control cell lines. Neither effect was seen in the N17ras transfectants, indicating that TGFbeta1 modulates integrin beta1-chain maturation by activating ras proteins. Cell fractionation studies demonstrated that this conversion takes place within the Golgi.

Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin.

Tyrosine phosphorylation of paxillin by the focal adhesion kinase (FAK) has been implicated as a signal transduction mechanism associated with cell adhesion and cytoskeletal reorganization. The potential role of serine phosphorylation of paxillin in these events has not been well characterized. In this study we have examined the phosphorylation profile of paxillin both in vitro and in vivo. By using glutathione S-transferase-paxillin fusion proteins in precipitation-kinase assays in vitro we observed that a fusion protein spanning amino acid residues 54-313 of paxillin, and containing a FAK-binding site, precipitated substantial serine kinase activity as well as FAK activity from a smooth-muscle lysate. Together these kinases phosphorylated paxillin on tyrosine residue 118, a site that has been identified previously as a target for FAK phosphorylation, and on serine residues 188 and/or 190. The binding site for the serine kinase, the identity of which is currently unknown, was further mapped to residues 168-191 of paxillin. To assess the physiological relevance of these sites phosphorylated in vitro, the profile of paxillin phosphorylation in vivo stimulated by seeding fibroblasts on fibronectin was characterized. As expected, plating cells on fibronectin enhanced the tyrosine phosphorylation of paxillin. However, 96% of the phosphorylation of paxillin occurred on serine residues. Comparison by two-dimensional phosphopeptide analyses indicated that the major sites of tyrosine and serine phosphorylation detected in the assays in vitro co-migrate with phosphopeptides derived from paxillin phosphorylated in vivo in response to plating cells on fibronectin. These findings support a role for both tyrosine and serine kinases in the signal transduction pathway linking integrin activation to paxillin phosphorylation.

Signal transduction for taurocholic acid in the olfactory system of Atlantic salmon.

Conjugated bile acids such as taurocholic acid (TChA) are potent olfactory stimuli for Atlantic salmon (Salmo salar). A plasma membrane rich fraction was derived from salmon olfactory rosettes and used to investigate TChA signal transduction and receptor binding. In the presence of GTP gamma S, TChA caused dose-dependent stimulation of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown, half maximal at less than 10(-7) M TChA. Stimulation of PIP2 breakdown by TChA required GTP gamma S, was blocked by GDP beta S, and was mimicked by A1F4-, consistent with a G protein requirement. A1F4- and Ca2+ stimulated breakdown of PIP2, but not phosphatidylcholine, arguing against a non-specific lipase activation. Stimulation of PIP2 breakdown by TChA was maximal at low Ca2+ concentration, < or = 10 nM. Conventional binding analysis with 3H-TChA was inconclusive due to a high degree of non-specific binding and to lack of tissue specificity expected for an olfactory receptor. Analysis of odorant amino acid binding indicated possible interaction of TChA with a putative acidic amino acid receptor but no interaction of TChA with a putative neutral amino acid receptor. We conclude that olfactory discrimination between amino acids and bile acids occurs in part at the receptor level while both classes of odors appear to use the same signal transduction mechanism, G protein mediated activation of phosphoinositide specific phospholipase C (PLC).