30 research outputs found
inhibition of osteoclast activity by complement regulation with df3016a a novel small molecular weight c5ar inhibitor
Abstract Recent insights have indicated an active role of the complex complement system not only in immunity, but also in bone remodeling. Evidence from knockout mice and observations from skeletal diseases have drawn attention to the C5a/C5aR axis of the complement cascade in the modulation of osteoclast functions and as potential therapeutic targets for treatment of bone pathologies. With the aim to identify novel C5aR regulators, a medicinal chemistry program was initiated, driven by structural information on a minor pocket of C5aR that has been proposed to be a key motif for C5aR intracellular activation. The impact of the peptidomimetic orthosteric C5aR antagonist (PMX-53), of two newly synthesized allosteric C5aR antagonists (DF2593A, DF3016A), and of C5aR down-regulation by specific siRNAs, were examined for regulation of osteoclastogenesis, using a well-validated in-vitro model starting from RAW264.7 precursor cells. Both pharmacological and molecular approaches reduced osteoclast maturation of RAW264.7 cells induced by receptor-activator of nuclear factor kappa-B ligand (RANKL), which limited the transcription of several differentiation markers evaluated by real-time PCR, including nuclear factor of activated T-cell 1, matrix metalloproteinase-9, cathepsin-K, and tartrate-resistant acid phosphatase. These treatments were ineffective on the subsequent step of osteoclast syncytium formation, apparently as a consequence of reduction of C5aR mRNA levels in the course of osteoclastogenesis, as monitored by real-time PCR. Among the C5aR antagonists analyzed, DF3016A inhibited osteoclast degradation activity through inhibition of C5aR signal transduction and transcription. These data confirm the preclinical relevance of this novel therapeutic candidate
Role of G Protein-coupled Receptor Kinase 4 and β-Arrestin 1 in Agonist-stimulated Metabotropic Glutamate Receptor 1 Internalization and Activation of Mitogen-activated Protein Kinases
The metabotropic glutamate 1 (mGlu(1)) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu(1) receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu(1) receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Galpha(q). In cells transfected with GRK4 and exposed to agonist, beta-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu(1) receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of beta-arrestin V53D dominant negative mutant, which did not affect the mGlu(1) receptor internalization, reduced by 70-80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu(1) receptor. The agonist-stimulated differential sorting of the mGlu(1) receptor and beta-arrestin as well as the activation of MAP kinases by mGlu(1) agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of beta-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing beta-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu(1) receptor by different mechanisms and that beta-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule
HSP 27 aspossible prognostic factor in patients with oral squamous cell carcinoma.
Summary. HSP27 belongs to the Heat shock protein
(HSP) family, which plays essential functions in cells
under physiological conditions and prevents stressinduced
cellular damage. The aim of this study was to
investigate the biological role of HSP27 in oral
tumorigenesis. Materials and methods: Seventy-nine
cases of oral squamous cell carcinoma and 10 cases of
normal mucosa were analysed for HSP27 expression by
immunohistochemistry. Moreover, the western blot
analysis was performed on two cases of normal mucosa
and five cases of OSCC. Results: Normal oral mucosa
showed a suprabasal expression of HSP27. Twenty-four
cases of SCC (30.7%) showed a diffuse staining for
HSP27, and 48 cases (60.3%) showed instead a decrease
in staining, which was diffuse, homogeneous, or with
alternation of positive and negative areas in a single
tumor (“mosaic” pattern). Only 7 cases of OSCC (7.5%)
were completely negative for HSP27. Frequency of
lymph node metastases was higher in HSP27-negative
tumours (3/7, 42.8%) than in HSP-reduced (16/48,
33.3%) or positive ones (5/26, 19.2%). Regard staging,
stages I and II had a higher score than stages III and IV
(stage I > stage II > stage III > stage IV). There was also
a statistically significant correlation between HSP27
expression and grade: HSP27 expression was reduced in
poorly differentiated tumours (P < 0.05). When analysed
for prognostic significance, patients with
negative/reduced HSP27 expression had poorer survival
rates than the group with positive HSP27 expression (P
< 0.05). The statistical analysis of these findings showed
no significant correlation between HSP27 expression,
sex, and tumour size. Conclusion: Cases with reduced
expression were more aggressive and poorly
differentiated. These data suggest that HSP27 expression may be useful in order to identify cases of oral squamous
cell carcinoma with more aggressive and invasive
phenotype providing novel diagnostic and prognostic
information on individual patient survival with oral
cancers
Is expression of p120ctn in oral squamous cell carcinomas a prognostic factor?
Objectives p120ctn is a component of the catenin family. To date, there have only been two studies examining expression levels of p120ctn in oral squamous cell carcinoma (OSCC). Materials and methods Paraffined specimens of 113 OSCCs and 12 of normal mucosa were examined by immunohistochemistry. Frozen samples of 20 OSCCs and 5 of normal mucosa were examined by Western blot (WB). Results were correlated with clinicopathological parameters. Five cell lines were examined by immunofluorescence, immunocytochemistry, and WB to show immunoreactivity and cellular localization of p120ctn. Results Altered p120ctn expression was observed in 109/113 cases of OSCC. Heterogenous cytoplasmic/nuclear expression was associated with loss of membranous distribution (88/113 cases). Complete loss of expression was noted in 21/113 cases. Increased cytoplasmic expression was evident in all positive cases, without significant correlation among p120ctn staining/pattern and grading/stage. Reduction/absence of p120ctn expression was related to poor prognosis ( P Conclusion p120ctn delocalization/loss of expression could be an independent prognostic marker in OSCC
60kDa Lysophospholipase, a New Sgk1 Molecular Partner Involved in the Regulation of ENaC
The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of ENaC-mediated sodium transport and is involved in the transduction of growth-factor-dependent cell survival and proliferation. The identification of molecular partners for Sgk1 is crucial for the understanding of its mechanisms of action. We performed a yeast two-hybrid screening based on a human kidney cDNA library to identify molecular partners of Sgk1. As a result the screening revealed a specific interaction between Sgk1 and a 60 kDa Lysophospholipase (LysoLP). LysoLP is a poorly characterized enzyme that, based on sequence analysis, might possess lysophospholipase and asparaginase activities. We demonstrate that LysoLP has indeed a lysophospholipase activity and affects metabolic functions related to cell proliferation and regulation of membrane channels. Moreover we demonstrate in the Xenopus oocyte expression system that LysoLP downregulates basal and Sgk1-dependent ENaC activity. In conclusion LysoLP may represent a new player in the regulation of ENaC and Sgk1-dependent signaling
Group IV Phospholipase A2α Controls the Formation of Inter-Cisternal Continuities Involved in Intra-Golgi Transport
The enzyme phospholipase A2 (cPLA2α) is involved in the formation of intercisternal tubules that mediate transport of proteins within the Golgi complex
The Glycerophosphoinositols: From Lipid Metabolites to Modulators of T-Cell Signaling
Glycerophosphoinositols (GPIs) are bioactive, diffusible phosphoinositide metabolites of
phospholipase A2 that act both intracellularly and in a paracrine fashion following their
uptake by specific transporters.The most representative compound, glycerophosphoinositol
(GroPIns), is a ubiquitous component of eukaryotic cells that participates in central
processes, including cell proliferation and survival. Moreover, glycerophosphoinositol 4-
phosphate (GroPIns4P) controls actin dynamics in several cell systems by regulating Rho
GTPases. Recently, immune cells have emerged as targets of the biological activities of
the GPIs. We have shown that exogenous GroPIns4P enhances CXCL12-induced T-cell
chemotaxis through activation of the kinase Lck in a cAMP/PKA-dependent manner. While
highlighting the potential of GroPIns4P as an immunomodulator, this finding raises questions
on the role of endogenously produced GroPIns4P as well as of other GPIs in the
regulation of the adaptive immune responses under homeostatic and pathological settings.
Here we will summarize our current understanding of the biological activities of the GPIs,
with a focus on lymphocytes, highlighting open questions and potential developments in
this promising new area
Regulation of G-protein-coupled receptor kinase subtypes by calcium sensor proteins
(GPCR) homologous desensitization is intrinsically related to the function of a class of S/T kinases named G-protein-coupled receptor kinases (GRK). GRK family is so far composed of six cloned members, named GRK1 to 6, which are classified into three subfamilies: GRK1 is alone in the first (rhodopsin kinase subfamily), GRK2 and 3 form the second [b-adrenergic receptor kinase (bARK) subfamily], and GRK4, 5, and 6 constitute the third (GRK4 subfamily). Recent studies from different laboratories have demonstrated that different calcium sensor proteins (CSP) can selectively regulate the activity of GRK subtypes. In the presence of calcium, rhodopsin kinase (GRK1) is inhibited by the photoreceptor-specific CSP recoverin through direct binding. Severa