Presence of substance P and the neurokinin-1 receptor in tenocytes of the human Achilles tendon

Nerve signal substances, such as the tachykinin substance P (SP), may be involved in the changes that occur in response to tendinopathy (tendinosis). It is previously known that the level of SP innervation within tendon tissue is limited, but results of experimental studies have suggested that SP may have stimulatory, angiogenetic and healing effects in injured tendons. Therefore, it would be of interest to know if there is a local SP-supply in tendon tissue. In the present study, the patterns of expression of SP and its preferred receptor, the neurokinin-1 receptor (NK-1 R), in normal and tendinosis human Achilles tendons were analyzed by use of both immunohistochemistry and in situ hybridization. We found that there was expression of SP mRNA in tenocytes, and that tenocytes showed expression of NK-1 R at protein as well as mRNA levels. The observations concerning both SP and NK-1 R were most evident for tenocytes in tendinosis tendons. Our findings suggest that SP is produced in tendinosis tendons, and furthermore that SP has marked effects on the tenocytes via the NK-1 R. It cannot be excluded that the SP effects are of importance concerning the processes of reorganization and healing that occur for tendon tissue in tendinosis. In conclusion, it appears as if SPergic autocrine/paracrine effects occur in tendon tissue during the processes of tendinosis, hitherto unknown effects for human tendons.     

Immunohistochemical and in situ hybridization observations favor a local catecholamine production in the human Achilles tendon

Results of recent studies using immunohistochemistry show evidence of an occurrence of catecholamine production in the cells (tenocytes) of patellar tendons exhibiting tendinopathy (tendinosis). In the present study, antibodies against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) and alpha1-adrenoreceptors were applied to sections of specimens of normal and tendinosis Achilles tendons. In situ hybridization using a probe detecting human TH mRNA was also utilized. It was found that sympathetic innervation was very scarce. On the other hand, there were distinct alpha1-adrenoreceptor immunoreactions in blood vessel walls. Interestingly, tenocytes, particularly from tendinosis samples in which the tenocytes showed an abnormal shape (not the typical slender appearance), displayed TH immunoreactions and reactions for TH mRNA. Of further interest was the finding of alpha1-adrenoreceptor immunoreactions in tenocytes. The observations show not only evidence of local catecholamine production at the protein level, which was the case in recent studies for the patellar tendon, but also at the mRNA level. The observations suggest that the tenocytes, especially those with disfigured appearances in tendinosis, can produce catecholamines and also that they can respond to sympathetic transmitters. This is of interest as adrenergic stimulation in other parts of the body is known to induce degenerative/apoptotic and proliferative events, features which are seen in Achilles tendinosis. These observations are completely new findings concerning the human Achilles tendon. It is likely that locally produced catecholamines and the occurrence of autocrine/paracrine effects of these substances are of great relevance during the process of tendinosis.     

Presence of a non-neuronal cholinergic system and occurrence of up- and down-regulation in expression of M2 muscarinic acetylcholine receptors: new aspects of importance regarding Achilles tendon tendinosis (tendinopathy)

Limited information is available concerning the existence of a cholinergic system in the human Achilles tendon. We have studied pain-free normal Achilles tendons and chronically painful Achilles tendinosis tendons with regard to immunohistochemical expression patterns of the M(2) muscarinic acetylcholine receptor (M(2)R), choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT). M(2)R immunoreactivity was detected in the walls of blood vessels. As evidenced via parallel staining for CD31 and alpha-smooth muscle actin, most M(2)R immunoreactivity was present in the endothelium. M(2)R immunoreactivity also occured in tenocytes, which regularly immunoreact for vimentin. The degree of M(2)R immunoreactivity was highly variable, tendinosis tendons that exhibit hypercellularity and hypervascularity showing the highest levels of immunostaining. Immunoreaction for ChAT and VAChT was detected in tenocytes in tendinosis specimens, particularly in aberrant cells. In situ hybridization revealed that mRNA for ChAT is present in tenocytes in tendinosis specimens. Our results suggest that autocrine/paracrine effects occur concerning the tenocytes in tendinosis. Up-regulation/down-regulation in the levels of M(2)R immunoreactivity possibly take place in tenocytes and blood vessel cells during the various stages of tendinosis. The presumed local production of acetylcholine (ACh), as evidenced by immunoreactivity for ChAT and VAChT and the detection of ChAT mRNA, appears to evolve in response to tendinosis. These observations are of importance because of the well-known vasoactive, trophic, and pain-modulating effects that ACh is known to have and do unexpectedly establish the presence of a non-neuronal cholinergic system in the Achilles tendon.     

Neuroprotection by NGF and BDNF Against Neurotoxin-Exerted Apoptotic Death in Neural Stem Cells Are Mediated Through Trk Receptors,Activating PI3-Kinase and MAPK Pathways

Neural stem cells (NSC) undergo apoptotic cell death during development of nervous system and in adult. However, little is known about the biochemical regulation of neuroprotection by neurotrophin in these cells. In this report, we demonstrate that Staurosporine (STS) and Etoposide (ETS) induced apoptotic cell death of NSC by a mechanism requiring Caspase 3 activation, poly (ADP-ribose) polymerase and Lamin A/C cleavage. Although C17.2 cells revealed higher mRNA level of p75 neurotrophin receptor (p75^NTR) compared with TrkA or TrkB receptor, neuroprotective effect of both nerve growth factor (NGF) and brain-derived growth factor (BDNF) mediated through the activation of tropomyosin receptor kinase (Trk) receptors. Moreover, both NGF and BDNF induced the activation of the phosphatidylinositide 3 kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathway. Inhibition of Trk receptor by K252a reduced PARP cleavage as well as cell viability, whereas inhibition of p75^NTR did not affect the effect of neurotrophin on neurotoxic insults. Thus our studies indicate that the protective effect of NGF and BDNF in NSC against apoptotic stimuli is mediated by the PI3K/Akt and MAPK signaling pathway via Trk receptors. An erratum to this article can be found at     

Functional analysis of mutant neurotrophins deficient in low-affinity binding reveals a role for p75LNGFR in NT-4 signalling.

The neurotrophins mediate their effects through binding to two classes of receptors, a tyrosine kinase receptor, member of the Trk family, and the low-affinity neurotrophin receptor, p75LNGFR, of as yet undefined signalling capacity. The need for a two-component receptor system in neurotrophin signalling is still not understood. Using site-directed mutagenesis, we have identified positively charged surfaces in BDNF, NT-3 and NT-4 that mediate binding to p75LNGFR. Arg31 and His33 in NT-3, and Arg34 and Arg36 in NT-4, located in an exposed hairpin loop, were found to be essential for binding to p75LNGFR. In BDNF, however, positively charged residues critical for p75LNGFR binding (Lys95, Lys96 and Arg97) were found in a spatially close but distinct loop region. Models of each neurotrophin were built using the coordinates of NGF. Analysis of their respective electrostatic surface potentials revealed similar clusters of positively charged residues in each neurotrophin but with differences in their precise spatial locations. Disruption of this positively charged interface abolished binding to p75LNGFR but not activation of cognate Trk receptors or biological activity in Trk-expressing fibroblasts. Unexpectedly, loss of low-affinity binding in NT-4, but not in BDNF or NT-3, affected receptor activation and biological activity in neuronal cells co-expressing p75LNGFR and TrkB, suggesting a role for p75LNGFR in regulating biological responsiveness to NT-4. These findings reveal a possible mechanism of ligand discrimination by p75LNGFR and suggest this receptor may selectively modulate the biological actions of specific neurotrophin family members.     

Role of endogenous brain-derived neurotrophic factor and sortilin in B cell survival

Brain-derived neurotrophic factor (BDNF), a major neuronal growth factor, is also known to exert an antiapoptotic effect in myeloma cells. Whereas BDNF secretion was described in B lymphocytes, the ability of B cells to produce sortilin, its transport protein, was not previously reported. We studied BDNF production and the expression of its receptors, tyrosine protein kinase receptor B and p75 neurotrophin receptor in the human pre-B, mature, and plasmacytic malignant B cell lines under normal and stress culture conditions (serum deprivation, Fas activation, or their combination). BDNF secretion was enhanced by serum deprivation and exerted an antiapoptotic effect, as demonstrated by neutralization experiments with antagonistic Ab. The precursor form, pro-BDNF, also secreted by B cells, decreases under stress conditions in contrast to BDNF production. Stress conditions induced the membranous expression of p75 neurotrophin receptor and tyrosine protein kinase receptor B, maximal in mature B cells, contrasting with the sequestration of both receptors in normal culture. By blocking Ab and small interfering RNA, we evidenced that BDNF production and its survival function are depending on sortilin, a protein regulating neurotrophin transport in neurons, which was not previously described in B cells. Therefore, in mature B cell lines, an autocrine BDNF production is up-regulated by stress culture conditions and exerts a modulation of apoptosis through the sortilin pathway. This could be of importance to elucidate certain drug resistances of malignant B cells. In addition, primary B lymphocytes contained sortilin and produced BDNF after mitogenic activation, which suggests that sortilin and BDNF might be implicated in the survival and activation of normal B cells also.     

Substance P is a mechanoresponsive, autocrine regulator of human tenocyte proliferation

It has been hypothesised that substance P (SP) may be produced by primary fibroblastic tendon cells (tenocytes), and that this production, together with the widespread distribution of the neurokinin-1 receptor (NK-1 R) in tendon tissue, could play an important role in the development of tendinopathy, a condition of chronic tendon pain and thickening. The aim of this study was to examine the possibility of endogenous SP production and the expression of NK-1 R by human tenocytes. Because tendinopathy is related to overload, and because the predominant tissue pathology (tendinosis) underlying early tendinopathy is characterized by tenocyte hypercellularity, the production of SP in response to loading/strain and the effects of exogenously administered SP on tenocyte proliferation were also studied. A cell culture model of primary human tendon cells was used. The vast majority of tendon cells were immunopositive for the tenocyte/fibroblast markers tenomodulin and vimentin, and immunocytochemical counterstaining revealed that positive immunoreactions for SP and NK-1 R were seen in a majority of these cells. Gene expression analyses showed that mechanical loading (strain) of tendon cell cultures using the FlexCell© technique significantly increased the mRNA levels of SP, whereas the expression of NK-1 R mRNA decreased in loaded as compared to unloaded tendon cells. Reduced NK-1 R protein was also observed, using Western blot, after exogenously administered SP at a concentration of 10⁻⁷ M. SP exposure furthermore resulted in increased cell metabolism, increased cell viability, and increased cell proliferation, all of which were found to be specifically mediated via the NK-1 R; this in turn involving a common mitogenic cell signalling pathway, namely phosphorylation of ERK1/2. This study indicates that SP, produced by tenocytes in response to mechanical loading, may regulate proliferation through an autocrine loop involving the NK-1 R.     

Human tenocytes are stimulated to proliferate by acetylcholine through an EGFR signalling pathway

Studies of human patellar and Achilles tendons have shown that primary tendon fibroblasts (tenocytes) not only have the capacity to produce acetylcholine (ACh) but also express muscarinic ACh receptors (mAChRs) through which ACh can exert its effects. In patients with tendinopathy (chronic tendon pain) with tendinosis, the tendon tissue is characterised by hypercellularity and angiogenesis, both of which might be influenced by ACh. In this study, we have tested the hypothesis that ACh increases the proliferation rate of tenocytes through mAChR stimulation and have examined whether this mechanism operates via the extracellular activation of the epidermal growth factor receptor (EGFR), as shown in other fibroblastic cells. By use of primary human tendon cell cultures, we identified cells expressing vimentin, tenomodulin and scleraxis and found that these cells also contained enzymes related to ACh synthesis and release (choline acetyltransferase and vesicular acetylcholine transporter). The cells furthermore expressed mAChRs of several subtypes. Exogenously administered ACh stimulated proliferation and increased the viability of tenocytes in vitro. When the cells were exposed to atropine (an mAChR antagonist) or the EGFR inhibitor AG1478, the proliferative effect of ACh decreased. Western blot revealed increased phosphorylation, after ACh stimulation, for both EGFR and the extracellular-signal-regulated kinases 1 and 2. Given that tenocytes have been shown to produce ACh and express mAChRs, this study provides evidence of a possible autocrine loop that might contribute to the hypercellularity seen in tendinosis tendon tissue.     

The p75 neurotrophin receptor

The low-affinity p75 molecule and trk tyrosine kinases serve as receptors for target-derived neurotrophins. While the mechanism by which receptor tyrosine kinases impart intracellular signaling has become well understood, the precise roles of the p75 receptor are not fully defined. The p75 neurotrophin receptor belongs to a family of transmembrane molecules which also serve as receptors for the tumor necrosis factor family of cytokines. Each receptor shares a common extracellular structure highlighted by conserved cysteine-rich repeats. Because NGF, BDNF, NT-3, and NT-4/5 bind to p75 with similar affinity, p75 may either act as a common subunit in a neurotrophin receptor complex with trk family members, or act by independent mechanisms to mediate biological actions of each neurotrophin. 1994 John Wiley & Sons, Inc.     

The interaction of neurotrophins with the p75NTR common neurotrophin receptor: a comprehensive molecular modeling study

Neurotrophins are a family of proteins with pleiotropic effects mediated by two distinct receptor types, namely the Trk family, and the common neurotrophin receptor p75NTR. Binding of four mammalian neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), to p75NTR is studied by molecular modeling based on X-ray structures of the neurotrophins and the extracellular domain of p55TNFR, a homologue of p75NTR. The model of neurotrophin/receptor interactions suggests that the receptor binding domains of neurotrophins (loops I and IV) are geometrically and electrostatically complementary to a putative binding site of p75NTR, formed by the second and part of the third cysteine-rich domains. Geometric match of neurotrophin/receptor binding domains in the complexes, as characterized by shape complementarity statistic Sc, is comparable to known protein/protein complexes. All charged residues within the loops I and IV of the neurotrophins, previously determined as being critical for p75NTR binding, directly participate in receptor binding in the framework of the model. Principal residues of the binding site of p75NTR include Asp47, Lys56, Asp75, Asp76, Asp88, and Glu89. The additional involvement of Arg80 and Glu53 is specific for NGF and BDNF, respectively, and Glu73 participates in binding with NT-3 and NT-4/5. Neurotrophins are likely to induce similar, but not identical, conformational changes within the p75NTR binding site.     

Down-regulation of brain-derived neurotrophic factor and its signaling components in the brain tissues of scrapie experimental animals

Prion is a unique nucleic acid-free pathogen that causes human and animal fatal neurodegenerative diseases. Brain-derived neurotrophic factor (BDNF) is a prototypic neurotrophin that helps to support the survival of existing neurons, and encourage the growth and differentiation of new neurons and synapses through axonal and dendritic sprouting. There are two distinct classes of glycosylated receptors, neurotrophin receptor p75 (p75NTR) and tropomyosin-related kinase (Trk), that can bind to BDNF. To obtain insights into the possible alterations of brain BDNF and its signaling pathway in prion disease, the levels of BDNF and several molecules in the BDNF pathway in the brain tissues of scrapie agents 263K-infected hamsters were separately evaluated. Western blots and/or immunohistochemical (IHC) assays revealed that BDNF, TrkB, GRB2 and p75NTR, were significantly downregulated in the brain tissues of scrapie-infected rodents at terminal stage. Double-stained immunofluorescent assay (IFA) demonstrated that BDNF and phospho-TrkB predominately expressed in neurons. Dynamic analyses of the brain samples collected at the different time-points during the incubation period illustrated continuous decreases of BDNF, TrkB, phospho-TrkB, GRB2 and p75NTR, which correlated well with neuron loss. However, these proteins remained almost unchanged in the prion infected cell line SMB-S15 compared with those of its normal cell line SMB-PS. These data suggest that the BDNF signaling pathway is severely hindered in the brains of prion disease, which may contribute, at least partially, to the neuron death.     

Bicaudal‐D1 regulates the intracellular sorting and signalling of neurotrophin receptors

We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor‐containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain‐derived neurotrophic factor (BDNF)‐activated TrkB and p75 neurotrophin receptor (p75^NTR) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co‐localisation of these neurotrophin receptors with retromer‐associated sorting nexin 1. The resulting re‐routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling‐competent TrkB isoforms and p75^NTR available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand‐activated receptors.     

Efficient expansion of mouse primary tenocytes using a novel collagen gel culture method

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell–cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.     

Overcoming the inhibitors of myelin with a novel neurotrophin strategy

Myelin inhibitors activate a p75(NTR)-dependent signaling cascade in neurons that not only inhibits axonal growth but also prevents neurotrophins (NT) from stimulating growth. Most intriguingly, in addition to Trk receptors, neurotrophins also bind to p75(NTR). We have designed a "mini-neurotrophin" called B(AG) to activate TrkB in the absence of p75(NTR) binding. We find that B(AG) is as effective as the natural TrkB ligands (brain-derived neurotrophic factor (BDNF) and NT-4) at promoting neurite outgrowth from cerebellar neurons. Furthermore, the neurite outgrowth responses stimulated by BDNF and B(AG) are inhibited by a common set of reagents, including the Trk receptor inhibitor K252a, as well as protein kinase A and phosphoinositide 3-kinase inhibitors. However, in contrast to BDNF, B(AG) promotes growth in the presence of a myelin inhibitor or when antibodies directly activate the p75(NTR) inhibitory pathway. On the basis of this observation, we postulated that the binding of BDNF to the p75(NTR) might compromise the ability of BDNF to stimulate neurite outgrowth in an inhibitory environment. To test this, we used NGF, and an NGF-derived peptide, to compete for the BDNF/p75(NTR) interaction; remarkably, in the presence of either agent, BDNF acquired the ability to promote neurite outgrowth in the presence of a myelin inhibitor. The data suggest that in an inhibitory environment, the BDNF/p75(NTR) interaction compromises regeneration. Agents that activate Trk receptors in the absence of p75(NTR) binding, or agents that inhibit neurotrophin/p75(NTR) binding, might therefore be better therapeutic candidates than neurotrophins.     

Role of BDNF in Central Motor Structures and Motor Diseases

Brain-derived neurotrophic factor (BDNF), belonging to the neurotrophic family of growth factors, has a widespread distribution in the central and peripheral nervous systems. In central motor structures including the motor cortex, cerebellum, basal ganglia, and spinal cord, BDNF exerts both neurotrophic and direct electrophysiological effects via a high-affinity tyrosine receptor kinase B receptor and a common low-affinity p75 neurotrophin receptor. The underlying signaling pathways mainly involve mitogen-activated protein kinase cascades, phosphatidylinositol 3-kinase pathway, and phospholipase C-γ pathway. The loss of BDNF usually leads to neurodegeneration in these motor centers and eventually results in several severe motor diseases, such as amyotrophic lateral sclerosis, spinocerebellar ataxias, Parkinson’s disease, Huntington’s disease, as well as vestibular syndrome. In this review, we summarize the recent understanding of functions of BDNF in motor structures and suggest that BDNF may be a potent candidate for the treatment of these neurodegenerative motor diseases.     

A role for p75 neurotrophin receptor in the control of apoptosis-driven hair follicle regression.

To examine the mechanisms that underlie the neurotrophin-induced, apoptosis-driven hair follicle involution (catagen), the expression and function of p75 neurotrophin receptor (p75NTR), which is implicated in apoptosis control, were studied during spontaneous catagen development in murine skin. By RT-PCR, high steady-state p75NTR mRNA skin levels were found during the anagen-catagen transition of the hair follicle. By immunohistochemistry, p75NTR alone was strongly expressed in TUNEL+/Bcl2- keratinocytes of the regressing outer root sheath, but both p75NTR and TrkB and/or TrkC were expressed by the nonregressing TUNEL-/Bcl2+ secondary hair germ keratinocytes. To determine whether p75NTR is functionally involved in catagen control, spontaneous catagen development was compared in vivo between p75NTR knockout (-/-) and wild-type mice. There was significant catagen retardation in p75NTR knockout mice as compared to wild-type controls (P<0.05). Instead, transgenic mice-overexpressing NGF (promoter: K14) showed substantial acceleration of catagen (P<0.001). Although NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) accelerated catagen in the organ-cultured skin of C57BL/6 mice, these neurotrophins failed to promote catagen development in the organ-cultured p75NTR null skin. These findings suggest that p75NTR signaling is involved in the control of kerotinocyte apoptosis during catagen and that pharmacological manipulation of p75NTR signaling may prove useful for the treatment of hair disorders that display premature entry into catagen.     

Antagonistic effects of TrkB and p75(NTR) on NMDA receptor currents in post-synaptic densities transplanted into Xenopus oocytes

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are essential regulators of synaptic function in the adult CNS. A TrkB-mediated effect at excitatory synapses is enhancement of NMDA receptor (NMDA-R)-mediated currents. Recently, opposing effects of TrkB and the pan-neurotrophin receptor p75(NTR) on long-term synaptic depression and long-term potentiation have been reported in the hippocampus. To further study the regulation of NMDA-Rs by neurotrophin receptors in their native protein environment, we micro-transplanted rat forebrain post-synaptic densities (PSDs) into Xenopus oocytes. One-minute incubations of oocytes with BDNF led to dual effects on NMDA-R currents: either TrkB-dependent potentiation or TrkB-independent inhibition were observed. Pro-nerve growth factor, a ligand for p75(NTR) but not for TrkB, produced a reversible, dose-dependent, TrkB-independent and p75(NTR)-dependent inhibition of NMDA-Rs. Fractionation experiments showed that p75(NTR) is highly enriched in the PSD protein fraction. Immunoprecipitation and pull-down experiments further revealed that p75(NTR) is a core component of the PSD, where it interacts with the PDZ3 domain of the scaffolding protein SAP90/PSD-95. Our data provide striking evidence for a rapid inhibitory effect of p75(NTR) on NMDA-R currents that antagonizes TrkB-mediated NMDA-R potentiation. These opposing mechanisms might be present in a large proportion of forebrain synapses and may contribute importantly to synaptic plasticity.     

Widespread Neurotrophin Receptor Expression in the Immune System and Other Nonneuronal Rat Tissues

Abstract: RNase protection analysis using p75, trk A, and trk B RNA probes was used to examine mRNA expression in rat tissues, with particular emphasis on the immune system. Every tissue examined, with the exception of postnatal day 0 spleen, expressed p75 mRNA. Trk A mRNA was observed in tissues previously reported to be negative for the trk A receptor, such as kidney, thymus, lymph node, muscle, and lung. Neuronal tissues expressed only the long form of trk A, whereas nonneuronal tissues expressed both trk A forms. Trk B mRNA was expressed by the same tissues as trk A, plus heart and spleen. Neuronal tissues expressed full-length and truncated trk B, whereas nonneuronal tissues only expressed truncated trk B. During development of the thymus p75 mRNA levels increased and trk A mRNA levels decreased. Similarly, for the spleen, p75 mRNA levels increased and those of trk B decreased during development. The expression of p75, trk A and trk B was localized primarily to the stroma of the thymus and spleen, but there was some expression by the splenocytes and thymocytes. The widespread expression of neurotrophin receptors in areas not known to be targets for neurotrophins suggests broader functions for neurotrophins outside of the nervous system.     

ATP induced brain-derived neurotrophic factor expression and release from osteoarthritis synovial fibroblasts is mediated by purinergic receptor P2X4

Brain-derived neurotrophic factor (BDNF), a neuromodulator involved in nociceptive hypersensitivity in the central nervous system, is also expressed in synoviocytes of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. We investigated the role of P2 purinoreceptors in the induction of BDNF expression in synovial fibroblasts (SF) of OA and RA patients. Cultured SF from patients with symptomatic knee OA and RA were stimulated with purinoreceptor agonists ATP, ADP, or UTP. The expression of BDNF mRNA was measured by quantitative TaqMan PCR. BDNF release into cell culture supernatants was monitored by ELISA. P2X4 expression in synovial tissue was detected by immunohistochemistry. Endogenous P2X4 expression was decreased by siRNA transfection before ATP stimulation. Kinase pathways were blocked before ATP stimulation. BDNF mRNA expression levels in OASF were increased 2 h and 5 h after ATP stimulation. Mean BDNF levels in cell culture supernatants of unstimulated OASF and RASF were 19 (±9) and 67 (±49) pg/ml, respectively. BDNF levels in SF supernatants were only elevated 5 h after ATP stimulation. BDNF mRNA expression in OASF was induced both by P2X receptor agonists ATP and ADP, but not by UTP, an agonist of P2Y purinergic receptors. The ATP-induced BDNF mRNA expression in OASF was decreased by siRNA-mediated reduction of endogenous P2X4 levels compared to scrambled controls. Inhibition of p38, but not p44/42 signalling reduced the ATP-mediated BDNF mRNA induction. Here we show a functional role of the purinergic receptor P2X4 and p38 kinase in the ATP-induced expression and release of the neurotrophin BDNF in SF.