Regulation of PDGF production and ERK activation by estrogen is associated with TSC2 gene expression

Mechanisms that regulate the growth response to estrogen (17beta-estradiol, E2) are poorly understood. Recently, loss of function of the tuberous sclerosis complex 2 (TSC2) gene has been associated with E2-related conditions that are characterized by benign cellular proliferation. We examined the growth response to E2 in vascular smooth muscle cells (VSMCs) that possess wild-type TSC2 and compared them with ELT-3 smooth muscle cells that do not express TSC2. In TSC2-expressing VSMCs, growth inhibition in response to E2 was associated with downregulation of platelet-derived growth factor (PDGF), PDGF receptor (PDGFR), and limited activation of extracellular signal-regulated kinase (ERK). In contrast, the growth-promoting effect of E2 in TSC2-null ELT-3 cells was associated with induction of PDGF, robust phosphorylation of PDGFR, and sustained activation of ERK. Furthermore, in ELT-3 cells, cellular growth and ERK activation by E2 were inhibited by the PDGFR inhibitor tyrphostin AG 17 and by PDGF-neutralizing antibody. These results demonstrate that autocrine production of PDGF and augmentation of the ERK pathway leads to estrogen-induced cellular proliferation in TSC2-null cells, a pathway that was downregulated in cells that express TSC2. Understanding the mechanisms that regulate the diverse responses to the steroid hormone estrogen could lead to novel approaches to the treatment of estrogen-related diseases that are characterized by aberrant cell proliferation.     

CC chemokine ligand 1 promotes recruitment of eosinophils but not Th2 cells during the development of allergic airways disease

One of the characteristic features of allergic asthma is recruitment of large numbers of inflammatory cells including eosinophils and Th2 lymphocytes to the lung. This influx of inflammatory cells is thought to be a controlled and coordinated process mediated by chemokines and their receptors. It is thought that distinct, differential expression of chemokine receptors allows selective migration of T cell subtypes in response to the chemokines that bind these receptors. Th2 cells preferentially express CCR8 and migrate selectively to its ligand, CC chemokine ligand (CCL)1. We studied the role of the CCR8 ligand, CCL1, in the specific recruitment of Th2 cells and eosinophils to the lung in a murine model of allergic airway disease. We have demonstrated for the first time that CCL1 is up-regulated in the lung following allergen challenge. Moreover, a neutralizing Ab to CCL1 reduced eosinophil migration to the lung, but had no effect on recruitment of Th2 cells following allergen challenge. In addition, there was no change in airway hyperresponsiveness or levels of Th2 cytokines. In a Th2 cell transfer system of pulmonary inflammation, anti-CCL1 also failed to affect recruitment of Th2 cells to the lung following allergen challenge. Significantly, intratracheal instillation of rCCL1 increased recruitment of eosinophils but not Th2 cells to the lung in allergen-sensitized and -challenged mice. In summary, our results indicate that CCL1 is important for the pulmonary recruitment of eosinophils, rather than allergen-specific Th2 cells, following allergen challenge.     

Thermally induced structural changes in glycinin,the 11S globulin of soya bean (Glycine max)--an in situ spectroscopic study

The thermal denaturation behaviour of glycinin solutions has been studied in situ as a function of ionic strength using various spectroscopic methods. Changes in secondary structure occurred at temperatures above 60 degrees C, well before the onset of gelation. Even after heating to 95 degrees C, much of the native beta-sheet structure of glycinin was retained, as indicated by the amide I peak maximum at 1635 cm(-1) in the Fourier transformed infrared (FT-IR) spectrum. This was accompanied by an increase in the 1625 cm(-1) band, indicative of the formation of intermolecular beta-sheet associated with protein aggregation. Nuclear magnetic resonance (NMR) spectroscopy confirmed the presence of highly mobile regions in glycinin comprising predominantly of Gln and Glu residues, corresponding to mobile regions previously identified by crystallographic studies. There was also evidence of a hydrogen-bonded structure within this mobile region, which may correspond to an alpha-helical region from Pro(256) to (or just before) Pro(269) in proglycinin. This structure disappeared at 95 degrees C, when heat-set gel formation occurred, as indicated by a sudden broadening and weakening of the NMR signal. Otherwise the NMR spectrum changed little during heating, emphasising the remarkable thermal stability of glycinin. It is proposed that during heating the core beta-barrel structure remains intact, but that the interface between the beta-domains melts, revealing hydrophobic faces which may then form new structures in a gel-network. As Cys(45), which forms the disulfide with Cys(12) linking the acidic and basic polypeptides, is found in this interface, such a rearrangement of the individual beta-domains could be accompanied by cleavage of this disulfide bond, as is observed experimentally. Such information contributes to our understanding the aggregative behaviour of proteins, and hence develops knowledge-based strategies for controlling and manipulating it.     

Fluid flow induced PGE2 release by bone cells is reduced by glycocalyx degradation whereas calcium signals are not

It has been hypothesized that bone cells have a hyaluronic acid (HA) rich glycocalyx (cell coat or pericellular matrix) and that this contributes to bone cell mechanotransduction via fluid flow. The glycocalyx of bone cells of the MC3T3-E1 osteoblastic cell line and the MLO-Y4 osteocytic cell line were characterized. Alcian blue staining and lectin binding experiments suggested that these cells have a glycocalyx rich in HA. Sulphated proteoglycans were not detected. Staining with hyaluronic acid binding protein and degradation by hyaluronidase confirmed that HA was a major component of the glycocalyx. We subjected cells, with and without hyaluronidase treatment, to oscillating fluid flow under standardized in vitro conditions. There was no effect of glycocalyx degradation on the intracellular calcium signal, in either cell type, in terms of the percentage of cells responding (40-80%) or the magnitude of the response (2-5 times baseline). However, a 4-fold fluid flow induced increase in PGE2 was eliminated by hyaluronidase pre-treatment in MLO-Y4 cells. We conclude that under these conditions the calcium and PGE2 responses occur via different pathways. An intact glycocalyx is not necessary in order to initiate a calcium signal in response to oscillating fluid flow. However, in osteocyte-like cells the PGE2 pathway is more dependent on mechanical signals transmitted through the glycocalyx.     

Annexin 1 regulates cell proliferation by disruption of cell morphology and inhibition of cyclin D1 expression through sustained activation of the ERK1/2 MAPK signal

Cellular proliferation is controlled by the integration and coordination of extracellular signals. This study explores the role of the protein annexin 1 (ANXA1) in the regulation of such events. We show that ANXA1 has a cell-type independent, anti-proliferative function through sustained activation of the ERK signaling cascade. Moreover, ANXA1 reduces proliferation by ERK-mediated disruption of the actin cytoskeleton and ablation of cyclin D1 protein expression and not by ERK-mediated induction of the cyclin-dependent kinase, CDK2, inhibitor p21(cip/waf). Finally, ANXA1 regulates the ERK pathway at a proximal location, by SH2 domain-independent association with the adapter protein Grb-2. In summary, overexpression of ANXA1 mediates the disruption of normal cell morphology and inhibits cyclin D1 expression, therefore reducing cell proliferation through proximal modulation of the ERK signal transduction pathway.     

A novel polypyrimidine tract-binding protein paralog expressed in smooth muscle cells

Polypyrimidine tract-binding protein (PTB) is an abundant widespread RNA-binding protein with roles in regulation of pre-mRNA alternative splicing and 3'-end processing, internal ribosomal entry site-driven translation, and mRNA localization. Tissue-restricted paralogs of PTB have previously been reported in neuronal and hematopoietic cells. These proteins are thought to replace many general functions of PTB, but to have some distinct activities, e.g. in the tissue-specific regulation of some alternative splicing events. We report the identification and characterization of a fourth rodent PTB paralog (smPTB) that is expressed at high levels in a number of smooth muscle tissues. Recombinant smPTB localized to the nucleus, bound to RNA, and was able to regulate alternative splicing. We suggest that replacement of PTB by smPTB might be important in controlling some pre-mRNA alternative splicing events.     

Protease inhibitors, part 13: Specific, weakly basic thrombin inhibitors incorporating sulfonyl dicyandiamide moieties in their structure

A series of compounds has been prepared by reaction of dicyandiamide with alkyl/arylsulfonyl halides as well as arylsulfonylisocyanates to locate a lead for obtaining weakly basic thrombin inhibitors with sulfonyldicyandiamide moieties as the S1 anchoring group. The detected lead was sulfanilyl-dicyandiamide (K1 of 3 microM against thrombin, and 15 microM against trypsin), which has been further derivatized at the 4-amino group by incorporating arylsulfonylureido as well as amino acyl/dipeptidyl groups protected at the amino terminal moiety with benzyloxycarbonyl or tosylureido moieties. The best compound obtained (ts-D-Phe-Pro-sulfanilyl-dicyandiamide) showed inhibition constants of 9 nM against thrombin and 1400 nM against trypsin. pKa measurements showed that the new derivatives reported here do indeed possess a reduced basicity, with the pKa of the modified guanidine moieties in the range 7.9-8.3 pKa units. Molecular mechanics calculations showed that the preferred tautomeric form of these compounds is of the type ArSO2N=C(NH2) NH-CN, probably allowing for the formation of favorable interaction between this new anchoring group and the active site amino acid residue Asp 189, critical for substrate/inhibitor binding to this type of serine protease. Thus, the main finding of the present paper is that the sulfonyldicyandiamide group may constitute an interesting alternative for obtaining weakly basic, potent thrombin inhibitors, which bind with less affinity to trypsin.     

Effects of cell swelling on intracellular calcium and membrane currents in bovine articular chondrocytes

Chondrocytes experience a dynamic extracellular osmotic environment during normal joint loading when fluid is forced from the matrix, increasing the local proteoglycan concentration and therefore the ionic strength and osmolarity. To exist in such a challenging environment, chondrocytes must possess mechanisms by which cell volume can be regulated. In this study, we investigated the ability of bovine articular chondrocytes (BAC) to regulate cell volume during a hypo-osmotic challenge. We also examined the effect of hypo-osmotic stress on early signaling events including [Ca2+](i) and membrane currents. Changes in cell volume were measured by monitoring the fluorescence of calcein-loaded cells. [Ca2+](i) was quantified using fura-2, and membrane currents were recorded using patch clamp. BAC exhibited regulated volume decrease (RVD) when exposed to hypo-osmotic saline which was inhibited by Gd3+. Swelling stimulated [Ca2+](i) transients in BAC which were dependent on swelling magnitude. Gd3+, zero [Ca2+](o), and thapsigargin all attenuated the [Ca2+](i) response, suggesting roles for Ca2+ influx through stretch activated channels, and Ca2+ release from intracellular stores. Inward and outward membrane currents significantly increased during cell swelling and were inhibited by Gd3+. These results indicate that RVD in BAC may involve [Ca2+](i) and ion channel activation, both of which play pivotal roles in RVD in other cell types. These signaling pathways are also similar to those activated in chondrocytes subjected to other biophysical signals. It is possible, then, that these signaling events may also be involved in a mechanism by which mechanical loads are transduced into appropriate cellular responses by chondrocytes.