Mark S. Fahey

Decreased expression of TGF‐β cell surface receptors during progression of human oral squamous cell carcinoma

This study examined the immunocytochemical expression of the transforming growth factor‐β (TGF‐β) isoforms TGF‐β1, TGF‐β2, and TGF‐β3, together with the TGF‐β cell surface receptors TβR‐I and TβR‐II, in patient‐matched tissue pairs of normal human oral epithelium, primary squamous cell carcinomas, and metastatic lymph node tumour deposits. There were no significant differences in the intensity of TGF‐β isoform specific staining between the normal oral epithelium, the primary tumours, and the lymph node metastases. By contrast, there was significantly less TβR‐II in the metastases than in the primary tumour and between the primary tumour and the normal oral epithelium. Similar trends were evident with TβR‐I, but not at a statistically significant level. This study also examined the structure of TβR‐I and TβR‐II in normal human oral keratinocytes in vitro and in 14 human oral carcinoma cell lines with known responses to TGF‐β1. No structural abnormalities of TβR‐II were present in the normal keratinocytes or in 13 of 14 malignant cell lines; in one line, there were both normal and mutant forms of TβR‐II, the latter being in the form of a frameshift mutation with the insertion of a single adenine base (bases 709–718, codons 125–128), predicting a truncated receptor having no kinase domain. No defects were present in TβR‐I. The structures of TβR‐I and TβR‐II did not correlate with growth inhibition by TGF‐β1. The data suggest that decreased expression of TGF‐β receptors, rather than structural defects of these genes, may be important in oral epithelial tumour progression. In order to examine the functional significance of a specific decrease in TβR‐II expression, a dominant‐negative TβR‐II construct (dnTβR‐II) was transfected into a human oral carcinoma cell line with a normal TGF‐β receptor profile and known to be markedly inhibited by TGF‐β1. In those clones that overexpressed the dnTβR‐II, growth inhibition and Smad binding activity were decreased, whilst the regulation of Fra‐1 and collagenase‐1 remained unchanged following treatment with TGF‐β1. The results demonstrate that a decrease in TβR‐II relative to TβR‐I leads to selective gene regulation with loss of growth inhibition but continued transcription of AP‐1‐dependent genes that are involved in the regulation of the extracellular matrix. Copyright

Human ProNGF: biological effects and binding profiles at TrkA, P75NTR and sortilin

Nerve growth factor (NGF) promotes cell survival via binding to the tyrosine kinase receptor A (TrkA). Its precursor, proNGF, binds to p75NTR and sortilin receptors to initiate apoptosis. Current disagreement exists over whether proNGF acts neurotrophically following binding to TrkA. As in Alzheimer’s disease the levels of proNGF increase and TrkA decrease, it is important to clarify the properties of proNGF. Here, wild‐type and cleavage‐resistant mutated forms (M) of proNGF were engineered and their binding characteristics determined. M‐proNGF and NGF bound to p75NTR with similar affinities, whilst M‐proNGF had a lower affinity than NGF for TrkA. M‐proNGF behaved neurotrophically, albeit less effectively than NGF. M‐proNGF addition resulted in phosphorylation of TrkA and ERK1/2, and in PC12 cells elicited neurite outgrowth and supported cell survival. Conversely, M‐proNGF addition to cultured cortical neurons initiated caspase 3 cleavage. Importantly, these biological effects were shown to be mediated by unprocessed M‐proNGF. Surprisingly, binding of the pro region alone to TrkA, at a site other than that of NGF, caused TrkA and ERK1/2 phosphorylation. Our data show that M‐proNGF stimulates TrkA to a lesser degree than NGF, suggesting that in Alzheimer brain the increased proNGF : NGF and p75NTR : TrkA ratios may permit apoptotic effects to predominate over neurotrophic effects.

TrkAd5: A Novel Therapeutic Agent for Treatment of Inflammatory Pain and Asthma

Elevated levels of nerve growth factor have been linked to the onset and persistence of many pain-related disorders and asthma. Described here are the design, expression, refolding, and purification of a monomeric (nonstrand-swapped) form of the binding domain of the nerve growth factor receptor, designated TrkAd5. We have shown that TrkAd5 produced recombinantly binds nerve growth factor with picomolar affinity. TrkAd5 has been characterized using a variety of biophysical and biochemical assays and is shown here to be stable in both plasma and urine. The palliative effects of TrkAd5 are demonstrated in animal models of inflammatory pain and allergic asthma. We conclude that TrkAd5 will prove effective in ameliorating both acute and chronic conditions where nerve growth factor acts as a mediator and suggest a role for its application in vivo as a novel therapeutic.

Dysregulation of autocrine TGF-beta isoform production and ligand responses in human tumour-derived and Ha-ras-transfected keratinocytes and fibroblasts.

This study examined the autocrine production of TGF-beta 1, -beta 2 and -beta 3 in culture supernatants from tumour-derived (H series, n = 7; BICR series, n = 5), Ha-ras-transfected (n = 4) and normal (n = 2) human keratinocytes using a sandwich enzyme-linked immunosorbent assay (ELISA). Detection limits were 39.0 pg ml-1 for TGF-beta 1, 78.0 pg ml-1 for TGF-beta 2 and 1.9 ng ml-1 for TGF-beta 3. Tumour-derived oral keratinocytes predominantly produced less TGF-beta 1 than normal oral epithelial cells; the expression of endogenous TGF-beta 2 was variable. In keratinocytes containing mutant Ha-ras, TGF-beta 1 production was enhanced and TGF-beta 2 was undetectable. TGF-beta 3 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) but the protein was not detected in conditioned media, most probably because of the low detection limits of the ELISA for this isoform. Neutralisation experiments indicated that the latent TGF-beta peptide was secreted in keratinocyte conditioned medium. Seven tumour-derived keratinocyte cell lines (H series) and fibroblasts separated from normal (n = 1) and tumour-derived (n = 2) keratinocyte cultures were examined for their response to exogenous TGF-beta 1, -beta 2 and -beta 3. Six of seven tumour-derived keratinocyte cell lines were inhibited by TGF-beta 1 and TGF-beta 2 (-beta 1 > -beta 2); one cell line was refractory to both TGF-beta 1 and TGF-beta 2. Keratinocytes were inhibited (4 of 7), stimulated (1 of 7) or failed to respond (2 of 7) to TGF-beta 3, TGF-beta 1, -beta 2 and -beta 3 stimulated both normal and tumour-associated fibroblasts, but the tumour-associated fibroblasts showed less response to the ligands than their normal counterparts following prolonged treatment with each isoform. The results demonstrate variable autocrine production of TGF-beta isoforms by malignant keratinocytes, with loss of TGF-beta 1 generally associated with the tumour-derived phenotype and modification of endogenous isoform production dependent on the genetic background of the tumour cells. Further, the variable response of the tumour-derived keratinocytes and contiguous fibroblasts to the TGF-beta isoforms suggests that dysregulation of TGF-beta autocrine and paracrine networks are common characteristics of squamous epithelial malignancy.

NGF receptor TrkAd5: Therapeutic agent and drug design target

Biochemical studies have shown that domain 5 of the TrkA (tropomyosin receptor kinase A) receptor is involved in the binding of NGF (nerve growth factor). Crystallographic studies have confirmed this, demonstrating that one homodimer of NGF binds to two TrkAd5 molecules. TrkAd5 has been made recombinantly in Escherichia coli, purified and shown to bind NGF with picomolar affinity. We have used the co-ordinates of the crystal structure of the NGF-TrkAd5 complex to screen approximately two million compounds in silico for the identification of small molecule agonists/antagonists. Selected hits were shown to be active in an in vitro ligand-binding assay; structure-activity relationships are now being investigated. In addition, TrkAd5 has been shown to be efficacious in preclinical models of inflammatory pain and asthma by the sequestration of excess levels of endogenous NGF, and therefore represents a novel therapeutic agent.

Activation of a synapse weakening pathway by human Val66 but not Met66 pro-brain-derived neurotrophic factor (proBDNF)

Graphical abstract

Design and nuclear magnetic resonance (NMR) structure determination of the second extracellular immunoglobulin tyrosine kinase A (TrkAIg2) domain construct for binding site elucidation in drug discovery

The tyrosine kinase A (TrkA) receptor is a validated therapeutic intervention point for a wide range of conditions. TrkA activation by nerve growth factor (NGF) binding the second extracellular immunoglobulin (TrkAIg2) domain triggers intracellular signaling cascades. In the periphery, this promotes the pain phenotype and, in the brain, cell survival or differentiation. Reproducible structural information and detailed validation of protein–ligand interactions aid drug discovery. However, the isolated TrkAIg2 domain crystallizes as a β-strand-swapped dimer in the absence of NGF, occluding the binding surface. Here we report the design and structural validation by nuclear magnetic resonance spectroscopy of the first stable, biologically active construct of the TrkAIg2 domain for binding site confirmation. Our structure closely mimics the wild-type fold of TrkAIg2 in complex with NGF (1WWW.pdb), and the 1H–15N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution.

Towards a model of sporadic Alzheimer’s disease: aiding design of small molecule TrkA agonists

It appears that Alzheimer’s disease is a good deal more complete than previously thought and may well have multiple dimensions than just Amyloid Cascade Hypothesis as the only culprit causing the disease. DR. Kokjohn spoke about a more holistic approach taken in the research, where multiple factors are taken into effect in the cause of Alzheimer’s. Tyler explained the difference between Alzheimer’s & Dementia.