Nedd4-2functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl– channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 μg/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 μg/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.

Crucial roles for olfactory ensheathing cells and olfactory mucosal cells in the repair of damaged neural tracts

Olfactory ensheathing cells, the glial cells of the olfactory nervous system, exhibit unique growth-promoting and migratory properties that make them interesting candidates for cell therapies targeting neuronal injuries such as spinal cord injury. Transplantation of olfactory cells is feasible and safe in humans; however, functional outcomes are highly variable with some studies showing dramatic improvements and some no improvements at all. We propose that the reason for this is that the identity and purity of the cells is different in each individual study. We have shown that olfactory ensheathing cells are not a uniform cell population and that individual subpopulations of OECs are present in different regions of the olfactory nervous system, with strikingly different behaviors. Furthermore, the presence of fibroblasts and other cell types in the transplant can dramatically alter the behavior of the transplanted glial cells. Thus, a thorough characterization of the differences between olfactory ensheathing cell subpopulations and how the behavior of these cells is affected by the presence of other cell types is highly warranted.

Phagocytosis of bacteria by olfactory ensheathing cells and Schwann cells

Opportunistic bacterial infections of the nasal cavity could potentially lead to infection of the brain if the olfactory or trigeminal nerves are colonised. The olfactory nerve may be a more susceptible route because primary olfactory neurons are in direct contact with the external environment. Peripheral glia are known to be able to phagocytose some species of bacteria and may therefore provide a defence mechanism against bacterial infection. As the nasal cavity is frequently exposed to bacterial infections, we hypothesised that the olfactory and trigeminal nerves within the nasal cavity could be subjected to bacterial colonisation and that the olfactory ensheathing cells and Schwann cells may be involved in responding to the bacterial invasion. We have examined the ability of mouse OECs and Schwann cells from the trigeminal nerve and dorsal root ganglia to phagocytose Escherichia coli and Burkholderia thailandensis in vitro. We found that all three sources of glia were equally able to phagocytose E. coli with 75-85% of glia having phagocytosed bacteria within 24h. We also show that human OECs phagocytosed E. coli. In contrast, the mouse OECs and Schwann cells had little capacity to phagocytose B. thailandensis. Thus subtypes of peripheral glia have similar capacities for phagocytosis of bacteria but show selective capacity for the two different species of bacteria that were examined. These results have implications for the understanding of the mechanisms of bacterial infections as well as for the use of glia for neural repair therapies.

Spatial and developmental heterogeneity of calcium signaling in olfactory ensheathing cells

Olfactory ensheathing cells (OECs) are specialized glial cells in the mammalian olfactory system supporting growth of axons from the olfactory epithelium into the olfactory bulb. OECs in the olfactory bulb can be subdivided into OECs of the outer nerve layer and the inner nerve layer according to the expression of marker proteins and their location in the nerve layer. In the present study, we have used confocal calcium imaging of OECs in acute mouse brain slices and olfactory bulbs in toto to investigate physiological differences between OEC subpopulations. OECs in the outer nerve layer, but not the inner nerve layer, responded to glutamate, ATP, serotonin, dopamine, carbachol, and phenylephrine with increases in the cytosolic calcium concentration. The calcium responses consisted of a transient and a tonic component, the latter being mediated by store-operated calcium entry. Calcium measurements in OECs during the first three postnatal weeks revealed a downregulation of mGluR(1) and P2Y(1) receptor-mediated calcium signaling within the first 2 weeks, suggesting that the expression of these receptors is developmentally controlled. In addition, electrical stimulation of sensory axons evoked calcium signaling via mGluR(1) and P2Y(1) only in outer nerve layer OECs. Downregulation of the receptor-mediated calcium responses in postnatal animals is reflected by a decrease in amplitude of stimulation-evoked calcium transients in OECs from postnatal days 3 to 21. In summary, the results presented reveal striking differences in receptor responses during development and in axon-OEC communication between the two subpopulations of OECs in the olfactory bulb.

Mesenchymal stromal cells and the repair of cartilage tissue

Articular cartilage has a limited intrinsic repair capacity, and thus defects are more likely to further degrade rather than undergo spontaneous self-repair. Whilst a number of surgical techniques have been developed to repair cartilage defects, their efficacy is generally poor and total joint replacement remains the gold standard, albeit last resort, treatment option. Cell-based therapies hold the greatest promise, as they appear uniquely capable of generating de novo cartilage tissue. Two approved therapies (ACI and MACI) are based on the premise that the transplantation of ex vivo expanded autologous chondrocyte populations, harvested from a non-load bearing region of the same joint, could be utilized to effectively regenerate cartilage tissue in the primary defect site. These therapeutic strategies are partially limited by our inability to harvest and expand adequate numbers of autologous chondrocytes that retain the appropriate phenotype. By contrast, the harvest and expansion of large numbers of mesenchymal stem/stromal cells (MSC) derived from tissues such as bone marrow and adipose is comparatively straightforward and has become routine in laboratories worldwide. Additionally, our understanding of the biochemical and biophysical signals required to drive the chondrogenic differentiation of MSC is rapidly increasing. It is conceivable that in the near future MSC expansion and differentiation technologies will offer a means to generate sufficient cell numbers, of an appropriate phenotype, for use in cartilage defect repair. In this chapter we review the relative potential of MSC and their likely contribution to cartilage regeneration.

methoxy-poly(ethylene glycol) modified poly(L-lactide) enhanced cell affinity of human bone marrow stromal cells by the upregulation of 1-cadherin and delta-2-catenin

Poly(l-lactide) (PLLA), a versatile biodegradable polymer, is one of the most commonly-used materials for tissue engineering applications. To improve cell affinity for PLLA, poly(ethylene glycol) (PEG) was used to develop diblock copolymers. Human bone marrow stromal cells (hBMSCs) were cultured on MPEG-b-PLLA copolymer films to determine the effects of modification on the attachment and proliferation of hBMSC. The mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analyzed using RT-qPCR to understand the underlying mechanisms. It was found that MPEG-b-PLLA copolymer films significantly improved cell adhesion, extension, and proliferation.This was found to be related to the significant upregulation of two adhesion genes, CDH1 and CTNND2, which encode 1-cadherin and delta-2-catenin, respectively, two key components for the cadherin-catenin complex. In summary, MPEG-b-PLLA copolymer surfaces improved initial cell adhesion by stimulation of adhesion molecule gene expression.

Mesenchymal stem cells, neural lineage potential, heparan sulfate proteoglycans and the matrix

Along with the tri-lineage of bone, cartilage and fat, human mesenchymal stem cells (hMSCs) retain neural lineage potential. Multiple factors have been described that influence lineage fate of hMSCs including the extracellular microenvironment or niche. The niche includes the extracellular matrix (ECM) providing structural composition, as well as other associated proteins and growth factors, which collectively influence hMSC stemness and lineage specification. As such, lineage specific differentiation of MSCs is mediated through interactions including cell–cell and cell–matrix, as well as through specific signalling pathways triggering downstream events. Proteoglycans (PGs) are ubiquitous within this microenvironment and can be localised to the cell surface or embedded within the ECM. In addition, the heparan sulfate (HS) and chondroitin sulfate (CS) families of PGs interact directly with a number of growth factors, signalling pathways and ECM components including FGFs, Wnts and fibronectin. With evidence supporting a role for HSPGs and CSPGs in the specification of hMSCs down the osteogenic, chondrogenic and adipogenic lineages, along with the localisation of PGs in development and regeneration, it is conceivable that these important proteins may also play a role in the differentiation of hMSCs toward the neuronal lineage. Here we summarise the current literature and highlight the potential for HSPG directed neural lineage fate specification in hMSCs, which may provide a new model for brain damage repair.

The heparan sulfate proteoglycan (HSPG) glypican-3 mediates commitment of MC3T3-E1 cells toward osteogenesis

Heparan sulfate (HS) sugar chains attached to core proteoglycans (PGs) termed HSPGs mediate an extensive range of cell-extracellular matrix (ECM) and growth factor interactions based upon their sulfation patterns. When compared with non-osteogenic (maintenance media) culture conditions, under established osteogenic culture conditions, MC3T3-E1 cells characteristically increase their osteogenic gene expression profile and switch their dominant fibroblast growth factor receptor (FGFR) from FGFR1 (0.5-fold decrease) to FGFR3 (1.5-fold increase). The change in FGFR expression profile of the osteogenic-committed cultures was reflected by their inability to sustain an FGF-2 stimulus, but respond to BMP-2 at day 14 of culture. The osteogenic cultures decreased their chondroitin and dermatan sulfate PGs (biglycan, decorin, and versican), but increased levels of the HS core protein gene expression, in particular glypican-3. Commitment and progress through osteogenesis is accompanied by changes in FGFR expression, decreased GAG initiation but increased N- and O-sulfation and reduced remodeling of the ECM (decreased heparanase expression) resulting in the production of homogenous (21 kDa) HS chain. With the HSPG glypican-3 expression strongly upregulated in these processes, siRNA was used to knockdown this gene to examine the effect on osteogenic commitment. Reduced glypican-3 abrogated the expression of Runx2, and thus differentiation. The reintroduction of this HSPG into Runx2-null cells allowed osteogenesis to proceed. These results demonstrate the dependence of osteogenesis on specific HS chains, in particular those associated with glypican-3.

Sulfated glycosaminoglycans mediate the effects of FGF2 on the osteogenic potential of rat calvarial osteoprogenitor cells

Fibroblast growth factor-2 (FGF2) is a powerful promoter of bone growth. We demonstrate here that brief exposure to FGF2 enhances mineralized nodule formation in cultured rat osteoprogenitor cells due to an expansion of cells that subsequently mineralize. This mitogenic effect is mediated via sulfated glycosaminoglycans (GAGs), FGFR1, and the extracellular signal-regulated kinase (ERK) pathway. The GAGs involved in this stimulation are chondroitin sulfates (CS) rather than heparan sulfates (HS). However, continuous FGF2 treatment reduces alkaline phosphatase (ALP) activity, downregulates collagen Ialpha1 (ColIalpha1) and FGFR3 expression, upregulates the expression and secretion of osteopontin (OPN) and inhibits mineralization. The inhibitory effects of FGF2 on FGFR3 expression and ALP activity are also mediated by the ERK pathway, although the effects of FGF2 on ColIalpha1 and OPN expression are mediated by GAGs and PKC activity. Thus short-term activation of FGF2/FGFR1 promotes osteoprogenitor proliferation and subsequent differentiation, while long-term activation of FGF2 signaling disrupts mineralization by modulating osteogenic marker expression. This study thus establishes the central role of sulfated GAGs in the osteogenic progression of osteoprogenitors.

Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes

Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

Loss of chromosomal integrity in human mammary epithelial cells subsequent to escape from senescence

The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed.

Osteogenic differentiation of murine embryonic stem cells is mediated by fibroblast growth factor receptors

The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be fully elucidated. However, it has become clear that the family of fibroblast growth factors (FGFs) are centrally involved. In this study we examined the role of the FGF receptors (FGFRs 1-4) during osteogenesis in murine ES cells. Single cells were obtained after the formation of embryoid bodies, cultured on gelatin-coated plates, and coaxed to differentiate along the osteogenic lineage. Upregulation of genes was analyzed at both the transcript and protein levels using gene array, relative-quantitative PCR (RQ-PCR), and Western blotting. Deposition of a mineralized matrix was evaluated with Alizarin Red staining. An FGFR1-specific antibody was generated and used to block FGFR1 activity in mES cells during osteogenic differentiation. Upon induction of osteogenic differentiation in mES cells, all four FGFRs were clearly upregulated at both the transcript and protein levels with a number of genes known to be involved in osteogenic differentiation including bone morphogenetic proteins (BMPs), collagen I, and Runx2. Cells were also capable of depositing a mineralized matrix, confirming the commitment of these cells to the osteogenic lineage. When FGFR1 activity was blocked, a reduction in cell proliferation and a coincident upregulation of Runx2 with enhanced mineralization of cultures was observed. These results indicate that FGFRs play critical roles in cell recruitment and differentiation during the process of osteogenesis in mES cells. In particular, the data indicate that FGFR1 plays a pivotal role in osteoblast lineage determination.

Colonizing while migrating : how do individual enteric neural crest cells behave?

Background Directed cell migration is essential for normal development. In most of the migratory cell populations that have been analysed in detail to date, all of the cells migrate as a collective from one location to another. However, there are also migratory cell populations that must populate the areas through which they migrate, and thus some cells get left behind while others advance. Very little is known about how individual cells behave to achieve concomitant directional migration and population of the migratory route. We examined the behavior of enteric neural crest-derived cells (ENCCs), which must both advance caudally to reach the anal end and populate each gut region. Results The behaviour of individual ENCCs was examined using live imaging and mice in which ENCCs express a photoconvertible protein. We show that individual ENCCs exhibit very variable directionalities and speed; as the migratory wavefront of ENCCs advances caudally, each gut region is populated primarily by some ENCCs migrating non-directionally. After populating each region, ENCCs remain migratory for at least 24 hours. Endothelin receptor type B (EDNRB) signaling is known to be essential for the normal advance of the ENCC population. We now show that perturbation of EDNRB principally affects individual ENCC speed rather than directionality. The trajectories of solitary ENCCs, which occur transiently at the wavefront, were consistent with an unbiased random walk and so cell-cell contact is essential for directional migration. ENCCs migrate in close association with neurites. We showed that although ENCCs often use neurites as substrates, ENCCs lead the way, neurites are not required for chain formation and neurite growth is more directional than the migration of ENCCs as a whole. Conclusions Each gut region is initially populated by sub-populations of ENCCs migrating non-directionally, rather than stopping. This might provide a mechanism for ensuring a uniform density of ENCCs along the growing gut.

Integrin αvβ3 mediates upregulation of epidermal growth-factor receptor expression and activity in human ovarian cancer cells

Upon overexpression of integrin αvβ3 and its engagement by vitronectin, we previously showed enhanced adhesion, proliferation, and motility of human ovarian cancer cells. By studying differential expression of genes possibly related to these tumor biological events, we identified the epidermal growth-factor receptor (EGF-R) to be under control of αvβ3 expression levels. Thus in the present study we characterized αvβ3-dependent changes of EGF-R and found significant upregulation of its expression and activity which was reflected by prominent changes of EGF-R promoter activity. Upon disruption of DNA-binding motifs for the transcription factors p53, ETF, the repressor ETR, p50, and c-rel, respectively, we sought to identify DNA elements contributing to αvβ3-mediated EGF-R promoter induction. Both, the p53- and ETF-mutant, while exhibiting considerably lower EGF-R promoter activity than the wild type promoter, retained inducibility by αvβ3. Mutation of the repressor motif ETR, as expected, enhanced EGF-R promoter activity with a further moderate increase upon αvβ3 elevation. The p50-mutant displayed EGF-R promoter activity almost comparable to that of the wild type promoter with no impairment of induction by αvβ3. However, the activity of an EGF-R promoter mutant displaying a disrupted c-rel-binding motif did not only prominently decline, but, moreover, was not longer responsive to enhanced αvβ3, involving this DNA element in αvβ3-dependent EGF-R upregulation. Moreover, αvβ3 did not only increase the EGF-R but, moreover, also led to obvious co-clustering on the cancer cell surface. By studying αvβ3/EGF-R-effects on the focal adhesion kinase (FAK) and the mitogen activated protein kinases (MAPK) p44/42 (erk−1/erk−2), having important functions in synergistic crosstalk between integrins and growth-factor receptors, we found for both significant enhancement of expression and activity upon αvβ3/VN interaction and cell stimulation by EGF. Upregulation of the EGF-R by integrin αvβ3, both receptor molecules with a well-defined role as targets for cancer treatment, might represent an additional mechanism to adapt synergistic receptor signaling and crosstalk in response to an altered tumor cell microenvironment during ovarian cancer progression.

Stimulation of osteogenic and angiogenic ability of cells on polymers by pulsed laser deposition of uniform akermanite-glass nanolayer

Polymer biomaterials have been widely used for bone replacement/regeneration because of their unique mechanical properties and workability. Their inherent low bioactivity makes them lack osseointegration with host bone tissue. For this reason, bioactive inorganic particles have been always incorporated into the matrix of polymers to improve their bioactivity. However, mixing inorganic particles with polymers always results in inhomogeneity of particle distribution in polymer matrix with limited bioactivity. This study sets out to apply the pulsed laser deposition (PLD) technique to prepare uniform akermanite (Ca2MgSi2O7, AKT) glass nanocoatings on the surface of two polymers (non-degradable polysulfone (PSU) and degradable polylactic acid (PDLLA)) in order to improve their surface osteogenic and angiogenic activity. The results show that a uniform nanolayer composed of amorphous AKT particles (∼30nm) of thickness 130nm forms on the surface of both PSU and PDLLA films with the PLD technique. The prepared AKT-PSU and AKT-PDLLA films significantly improved the surface roughness, hydrophilicity, hardness and apatite mineralization, compared with pure PSU and PDLLA, respectively. The prepared AKT nanocoatings distinctively enhance the alkaline phosphate (ALP) activity and bone-related gene expression (ALP, OCN, OPN and Col I) of bone-forming cells on both PSU and PDLLA films. Furthermore, AKT nanocoatings on two polymers improve the attachment, proliferation, VEGF secretion and expression of proangiogenic factors and their receptors of human umbilical vein endothelial cells (HUVEC). The results suggest that PLD-prepared bioceramic nanocoatings are very useful for enhancing the physicochemical, osteogenic and angiogenic properties of both degradable and non-degradable polymers for application in bone replacement/regeneration.