10 research outputs found
Rab3D a regulator of exocytosis in non-neuronal cells
Rab3D, a small Ras-like GTPase, is a key
regulator of intracellular vesicle transport during
exocytosis. It has been shown that Rab3 GTPases are
abundant in cells with regulated secretory pathways and
are thought to confer the specificity of docking and
fusion during regulated exocytosis. Unlike other Rab3
isoforms, Rab3D is enriched in a number of nonneuronal
tissues and is localised to secretory granules in
the cytoplasm of these cells. The structure of Rab3D
exhibits all of the conserved domains from the Rab
family and also contains hypervariable N- and Cterminal
regions. Rab3D undergoes post-translational
isoprenylation and cycles between GDP- and GTPbound
forms. Apart from the factors involved in the Rab
activation cycle, few Rab3D effector proteins have been
identified to date. Nevertheless, it has long been
suggested that Rab3D plays a role in regulated
exocytotic processes as well as apically directed
transcytosis. This review summarises the recent work on
the biological function, structural integrity and
molecular interactions of Rab3D in non-neuronal cells
Structure and function of V-ATPases in osteoclasts: potential therapeutic targets for the treatment of osteolysis
Excessive activity of osteoclasts becomes
manifest in many common lytic bone disorders such as
osteoporosis, Paget’s disease, bone aseptic loosening and
tumor-induced bone destruction. Vacuolar proton pump
H+-adenosine triphosphatases (V-ATPases), located on
the bone-apposed plasma membrane of the osteoclast,
are imperative for the function of osteoclasts, and thus
are a potential molecular target for the development of
novel anti-resorptive agents. To date, the V-ATPases
core structure has been well modeled and consists of two
distinct functional domains, the V1 (A, B1, B2, C1, C2,
D, E1, E2, F, G1, G2, G3, and H subunits) and V0 (a1,
a2, a3, a4, d1, d2, c, c’ e1, e2 subunits) as well as the
accessory subunits ac45 and M8-9. However, the exact
configuration of osteoclast specific V-ATPases remains
to be established. Inactivation of subunit a3 leads to
osteopetrosis in both mice and man because of nonfunctional
osteoclasts that are capable of acidifying the
extracellular resorption lacuna. On the other hand,
inactivation of subunits c, d1 and ac45 results in early
embryonic lethality, indicating that certain subunits, such
as a3, are more specific to osteoclast function than
others. In osteoclasts, V-ATPases also cooperate with
chloride channel protein CLC-7 to acidify the resorption
lacuna. In addition, development of V-ATPases
inhibitors such as bafilomycin A1, SB 242784 and
FR167356 that selectively target osteoclast specific VATPases
remains a challenge. Understanding the subunits of V-ATPase regulate osteoclast function might
facilitate the development of novel and selective
inhibitors for the treatment of lytic bone disorders. This
review summarizes recent research developments in VATPases
with particular emphasis on osteoclast biology
The SQSTM1/p62 UBA domain regulates Ajuba localisation, degradation and NF-κB signalling function
The LIM-domain containing protein Ajuba and the scaffold protein SQSTM1/p62 regulate signalling of NF-κB, a transcription factor involved in osteoclast differentiation and survival. The ubiquitin-associated domain of SQSTM1/p62 is frequently mutated in patients with Paget’s disease of bone. Here, we report that Ajuba activates NF-κB activity in HEK293 cells, and that co-expression with SQSTM1/p62 inhibits this activation in an UBA domain-dependent manner. SQSTM1/p62 regulates proteins by targeting them to the ubiquitin-proteasome system or the autophagy-lysosome pathway. We show that Ajuba is degraded by autophagy, however co-expression with SQSTM1/p62 (wild type or UBA-deficient) protects Ajuba levels both in cells undergoing autophagy and those exposed to proteasomal stress. Additionally, in unstressed cells co-expression of SQSTM1/p62 reduces the amount of Ajuba present in the nucleus. SQSTM1/p62 with an intact ubiquitin-associated domain forms holding complexes with Ajuba that are not destined for degradation yet inhibit signalling. Thus, in situations with altered levels and localization of SQSTM1/p62 expression, such as osteoclasts in Paget’s disease of bone and various cancers, SQSTM1/p62 may compartmentalize Ajuba and thereby impact its cellular functions and disease pathogenesis. In Paget’s, ubiquitin-associated domain mutations may lead to increased or prolonged Ajuba-induced NF-κB signalling leading to increased osteoclastogenesis. In cancer, Ajuba expression promotes cell survival. The increased levels of SQSTM1/p62 observed in cancer may enhance Ajuba-mediated cancer cell survival
Evidence that human cartilage and chondrocytes do not express calcitonin receptor
Objective
Calcitonin (CT) has been recently shown to exhibit direct protective effects on articular cartilage against joint degenerative disease. It has been proposed that CT might act via the CT receptor (CTR) to activate the cyclic AMP (cAMP) pathway and protect type II collagen degradation. In this study, we investigated the existence of CTR in human articular cartilage and chondrocytes, and examined the potential pharmacological effects and transduction pathway of salmon CT (sCT) in human chondrocytes.
Methods
Five human articular cartilage samples were examined for the expression of the CTR by polymerase chain reaction (PCR), immunostaining and Western blot analysis. cAMP levels in human chondrocyte stimulated with sCT were assessed by ELISA. The effect of sCT on the gene expression profiles, including aggrecan, type II collagen, MMP-1, MMP-3 and MMP-13, of human chondrocytes was also examined by relative quantitative Real-time PCR.
Results
We failed to detect the CTR at both the transcriptional and protein levels in human chondrocytes and cartilage tissue by PCR, immunostaining and Western blotting. cAMP levels were significantly elevated in human chondrocytes by forskolin (100 μM) to more than 10-fold (P < 0.001), however, were not induced by sCT (10−7 M, 10−8 M, 10−9 M). Real-time PCR analysis demonstrated that sCT slightly reduced the gene expression of MMPs, although this effect was not statistically significant.
Conclusion
In contrary to previous reports, our data indicate that human cartilage and chondrocytes do not express CTR. Furthermore, sCT does not appear to have direct effects on human chondrocytes. We propose that the chondroprotective effect of CT observed in vivo may be indirect via its impact on subchondral bone resorptive activity of osteoclasts
Pharmacogenomics of off-target adverse drug reactions
Off-target adverse drug reactions (ADRs) are associated with significant morbidity and costs to the healthcare system, and their occurrence is not predictable based on the known pharmacological action of the drug's therapeutic effect. Off-target ADRs may or may not be associated with immunological memory, although they can manifest with a variety of shared clinical features, including maculopapular exanthema, severe cutaneous adverse reactions (SCARs), angioedema, pruritus and bronchospasm. Discovery of specific genes associated with a particular ADR phenotype is a foundational component of clinical translation into screening programmes for their prevention. In this review, genetic associations of off-target drug-induced ADRs that have a clinical phenotype suggestive of an immunologically mediated process and their mechanisms are highlighted. A significant proportion of these reactions lack immunological memory and current data are informative for these ADRs with regard to disease pathophysiology, therapeutic targets and biomarkers which may identify patients at greatest risk. Although many serious delayed immune-mediated (IM)-ADRs show strong human leukocyte antigen associations, only a small subset have successfully been implemented in screening programmes. More recently, other factors, such as drug metabolism, have been shown to contribute to the risk of the IM-ADR. In the future, pharmacogenomic targets and an understanding of how they interact with drugs to cause ADRs will be applied to drug design and preclinical testing, and this will allow selection of optimal therapy to improve patient safety
Long-term exposure of mice to 890Â ppm atmospheric CO2 alters growth trajectories and elicits hyperactive behaviours in young adulthood
Abstract: Atmospheric carbon dioxide (CO2) levels are currently at 418 parts per million (ppm), and by 2100 may exceed 900 ppm. The biological effects of lifetime exposure to CO2 at these levels is unknown. Previously we have shown that mouse lung function is altered by long-term exposure to 890 ppm CO2. Here, we assess the broader systemic physiological responses to this exposure. Mice were exposed to either 460 or 890 ppm from preconception to 3 months of age, and assessed for effects on developmental, renal and osteological parameters. Locomotor, memory, learning and anxiety-like behaviours of the mice were also assessed. Exposure to 890 ppm CO2 increased birthweight, decreased female body weight after weaning, and, as young adults, resulted in reduced engagement in memory/learning tasks, and hyperactivity in both sexes in comparison to controls. There were no clear anxiety, learning or memory changes. Renal and osteological parameters were minimally affected. Overall, this study shows that exposure of mice to 890 ppm CO2 from preconception to young adulthood alters growth and some behaviours, with limited evidence of compensatory changes in acid–base balance. These findings highlight the potential for a direct effect of increased atmospheric CO2 on mammalian health outcomes. (Figure presented.). Key points: Long-term exposure to elevated levels of atmospheric CO2 is an uncontrolled experiment already underway. This is the first known study to assess non-respiratory physiological impacts of long-term (conception to young adulthood) exposure of mice to CO2 at levels that may arise in the atmosphere due to global emissions. Exposure to elevated CO2, in comparison to control mice, altered growth patterns in early life and resulted in hyperactive behaviours in young adulthood. Renal and bone parameters, which are important to balance acid–base levels to compensate for increased CO2 exposure, remained relatively unaffected. This work adds to the body of evidence regarding the effects of carbon emissions on mammalian health and highlights a potential future burden of disease
Bone allograft non-union is related to excessive osteoclastic bone resorption: A sheep model study
Using a sheep femoral allograft model we
have investigated the cellular and molecular mechanisms
associated with non-union of bone allografts.
Histomorphometric analysis revealed that allograft nonunions
featured both marked increases in osteoclast
(OC) numbers and total eroded bone surface as
compared to allografts wich had undergone direct union.
Three distinct cellular layers lying adjacent to the
allograft bone surface were identified in all non-union
cases. The outer or fibroblastic layer contained an
abundance of fibroblasts and connective tissue.
Circumscribing this layer was a band of synovial-like
cells consisting mainly of large spindle-shaped
mononuclear cells mixed with scattered round-shaped
mononuclear cells. The third layer, which was directly
juxtaposed to the allograft bone surface, consisted
predominantly of multinuclear OCs which were
positively identified by calcitonin receptor
immunohistochemistry. Interestingly, in-situ
hybridisation revealed that surrounding synovial-like
cells in non-union allografts, expressed abundant gene
transcripts for receptor activator NF-kB ligand
(RANKL), a membrane bound factor critical for both the
induction of OC activity and osteoclastogenesis. We
propose that excessive bone resorption by host OCs
contributes, at least partially, to the failure of bone
allografts. The production of RANKL by synovial-like fibroblasts may be the driving force responsible for the
elevated generation and activation of OCs. Based on
such evidence novel therapeutic strategies for the
treatment of non-union bone allografts using anti-bone
resorbing agents may be devised