Src and ADAM-17-Mediated Shedding of Transforming Growth Factor-alpha Is a Mechanism of Acute Resistance to TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) has emerged as a promising anticancer agent. However, resistance to TRAIL is likely to be a major problem, and sensitization of cancer cells to TRAIL may therefore be an important anticancer strategy. In this study, we examined the effect of the epidermal growth factor receptor (EGFR)tyrosine kinase inhibitor (TKI) gefitinib and a human epidermal receptor 2 (HER2)-TKI (M578440) on the sensitivity of human colorectal cancer (CRC) cell lines to recombinant human TRAIL (rhTRAIL). A synergistic interaction between rhTRAIL and gefitinib and rhTRAIL and M578440 was observed in both rhTRAIL-sensitive and resistant CRC cells. This synergy correlated with an increase in EGFR and HER2 activation after rhTRAIL treatment. Furthermore, treatment of CRC cells with rhTRAIL resulted in activation of the Src family kinases (SFK). Importantly, we found that rhTRAIL treatment induced shedding of transforming growth factor-alpha (TGF-alpha) that was dependent on SFK activity and the protease ADAM-17. Moreover, this shedding of TGF-alpha was critical for rhTRAIL-induced activation of EGFR. In support of this, SFK inhibitors and small interfering RNAs targeting ADAM-17 and TGF-alpha also sensitized CRC cells to rhTRAIL-mediated apoptosis. Taken together, our findings indicate that both rhTRAIL-sensitive and resistant CRC cells respond to rhTRAIL treatment by activating an EGFR/HER2-mediated survival response and that these cells can be sensitized to rhTRAIL using EGFR/HER2-targeted therapies. Furthermore, this acute response to rhTRAIL is regulated by SFK-mediated and ADAM-17-mediated shedding of TGF-alpha, such that targeting SFKs or inhibiting ADAM-17, in combination with rhTRAIL, may enhance the response of CRC tumors to rhTRAIL. [Cancer Res 2008;68(20):8312-21]

Neuropeptides Regulate Expression of Angiogenic Growth Factors in Human Dental Pulp Fibroblasts

Abstract
INTRODUCTION:
Neuropeptides play an important role in inflammation and repair and have been implicated in mediating angiogenesis. Pulp fibroblasts express neuropeptide receptors, and the aim of this research was to investigate whether neuropeptides could regulate angiogenic growth factor expression in vitro
METHODS:
An angiogenic array was used to determine the levels of 10 angiogenic growth factors expressed by human pulp fibroblasts.
RESULTS:
Pulp fibroblasts were shown to express angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin-binding epidermal growth factor, hepatocyte growth factor, leptin, platelet-derived growth factor, placental growth factor, and vascular endothelial growth factor. Furthermore, the neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide Y altered angiogenic growth factor expression in vitro.
CONCLUSIONS:
The regulation of angiogenic growth factor expression by neuropeptides suggests a novel role for neuropeptides in pulpal inflammation and repair.

The effect of osteogenic growth factors on bone growth into a ceramic filled defect around an implant

Currently available synthetic bone substitutes perform poorly compared to autograft. It is hoped that by adding osteogenic growth factors to the materials, new bone formation could be increased and the clinical outcome improved. In this study, IGF-1, bFGF and TGFbeta1, alone and in combination, were absorbed onto a carrier of P-tricalcium phosphate (PTCP) and implanted into a defect around a hydroxyapatite-coated, stainless steel implant in the proximal tibia of rat in a model of revision arthroplasty. Animals were sacrificed at 6 and 26 weeks for routine histology and histomorphometry and mechanical push out tests. The results show that only bFGF had a significant effect on ceramic resorption. The groups that received bFGF and bFGF in combination with TGFbeta1 had smaller and fewer betaTCP particles remaining in the defect at 6 and 26 weeks. No growth factor combination significantly enhanced new bone formation or the mechanical strength of the implant. These results indicate that, of the growth factors tested, only bFGF had any beneficial effect on the host response to the implant, perhaps by delaying osteoblast differentiation and thereby prolonging osteoclast access to the ceramic. (C) 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Connective tissue growth factor antagonizes transforming growth factor-beta 1/Smad signalling in renal mesangial cells

The critical involvement of TGF-beta 1 (transforming growth factor-beta 1) in DN (diabetic nephropathy) is well established. However, the role of CTGF (connective tissue growth factor) in regulating the complex interplay of TGF-beta 1 signalling networks is poorly understood. The purpose of the present study was to investigate co-operative signalling between CTGF and TGF-beta 1 and its physiological significance. CTGF was determined to bind directly to the T beta RIII (TGF-beta type III receptor) and antagonize TGF-beta 1-induced Smad phosphorylation and transcriptional responses via its N-terminal half. Furthermore, TGF-beta 1 binding to its receptor was inhibited by CTGF. A consequent shift towards non-canonical TGF-beta 1 signalling and expression of a unique profile of differentially regulated genes was observed in CTGF/TGF-beta 1-treated mesangial cells. Decreased levels of Smad2/3 phosphorylation were evident in STZ (streptozotocin)-induced diabetic mice, concomitant with increased levels of CTGF Knockdown of T beta RIII restored TGF-beta 1-mediated Smad signalling and cell contractility, suggesting that T beta RIII is key for CTGF-mediated regulation of TGF-beta 1. Comparison of gene expression profiles from CTGF/TGF-beta 1-treated mesangial cells and human renal biopsy material with histological diagnosis of DN revealed significant correlation among gene clusters. In summary, mesangial cell responses to TGF-beta 1 are regulated by cross-talk with CTGF, emphasizing the potential utility of targeting CTGF in DN.

Gingival fibroblast response to cyclosporin A and transforming growth factor beta(1)

This study investigates a potential role for TGF beta(1), in the pathogenesis of cyclosporin A-induced gingival overgrowth (CsA-OG). TGF beta(1) was localized immunohistochemically in the connective tissue of both normal gingiva and CsA-OG. Intense staining for TGF beta(1) was detected at the tips of the dermal papillae of the overgrown gingiva. In addition, fibroblasts derived from healthy gingiva and fibroblasts derived from CsA-OG were cultured both as monolayers or embedded in a 3D-collagen gel. Fibroblast activity was monitored in terms of protein and collagen production in the presence of (i) 1 ng/ml TGF beta(1), (ii) 500 ng/ml CsA, or (iii) 500 ng/ml CsA and 1 ng/ml TGF beta(1). In monolayer culture TGF beta(1) significantly increased protein and collagen production in all cell strains (p

Role of vascular endothelial growth factor and placental growth factors during retinal vascular development and hyaloid regression

PURPOSE. Vascular endothelial growth factor (VEGF)-A and placental growth factor (PIGF) are members of a large group of homologous peptides identified as the VEGF family. Although VEGF-A is known to act as a potent angiogenic peptide in the retina, the vasoactive function of PIGF in this tissue is less well defined. This study has sought to elucidate the expression patterns and modulatory role of these growth factors during retinal vascular development and hyaloid regression in the neonatal mouse. METHODS. C57BL6J mice were killed at postnatal days (P)1, P3, P5, P7, P9, and P11. The eyes were enucleated and processed for in situ hybridization and immunocytochemistry and the retinas extracted for total protein or RNA. Separate groups of neonatal mice were also injected intraperitoneally daily from P2 through P9 with either VEGF-neutralizing antibody, PIGF-neutralizing antibody, isotype immunoglobulin (Ig)-G, or phosphate-buffered saline (PBS). The mice were then perfused with fluorescein isothiocyanate (FITC)-dextran, and the eyes were subsequently embedded in paraffin wax or flat mounted. RESULTS. Quantitative (real-time) reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar expression patterns of VEGF-A and PIGF mRNA during neonatal retinal development, although the fluctuation between time periods was greater overall for VEGF-A. The localization of VEGF-A and PIGF in the retina, as revealed by in situ hybridization and immunohistochemistry, was also similar. Neutralization of VEGF-A caused a significant reduction in the hyaloid and retinal vasculature, whereas PIGF antibody treatment caused a marked persistence of the hyaloid without significantly affecting retinal vascular development. CONCLUSIONS. Although having similar expression patterns in the retina, these growth factors appear to have distinct modulatory influences during normal retinal vascular development and hyaloid regression.

Endothelial lineage differentiation from induced pluripotent stem cells is regulated by microRNA-21 and transforming growth factor beta2 (TGF-β2) pathways

Finding a suitable cell source for endothelial cells (ECs) for cardiovascular regeneration is a challenging issue for regenerative medicine. In the paper we describe a novel mechanism regulating induced pluripotent stem cells (iPSC) differentiation into ECs, with a particular focus on miRNAs and their targets. We first established a protocol using collagen IV and VEGF to drive the functional differentiation of iPSCs into ECs and compared the miRNA signature of differentiated and undifferentiated cells. Among the miRNAs overrepresented in differentiated cells, we focused on microRNA-21 (miR-21) and studied its role in iPSC differentiation. Overexpression of miR-21 in pre-differentiated iPSCs induced EC marker upregulation and in vitro and in vivo capillary formation; accordingly, inhibition of miR-21 produced the opposite effects. Importantly, miR-21 overexpression increased TGF-β2 mRNA and secreted protein level, consistent with the strong upregulation of TGF-β2 during iPSC differentiation. Indeed, treatment of iPSCs with TGFβ-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGFβ-2, strongly decreased VE-cadherin expression. Furthermore, TGFβ-2 neutralization and knockdown inhibited miR-21-induced EC marker expression. Finally, we confirmed the PTEN/Akt pathway as a direct target of miR-21 and we showed that PTEN knockdown is required for miR-21 mediated endothelial differentiation. In conclusion, we elucidated a novel signaling pathway that promotes the differentiation of iPSC into functional ECs suitable for regenerative medicine applications.

Histone deacetylase 3 unconventional splicing mediates endothelial-to- mesenchymal transition through transforming growth factor β2

Histone deacetylase 3 (HDAC3) plays a critical role in the maintenance of endothelial integrity and other physiological processes. In this study, we demonstrated that HDAC3 undergoes unconventional splicing during stem cell differentiation. Four different splicing variants have been identified, designated as HD3α, -β, -γ, and -Δ, respectively. HD3α was confirmed in stem cell differentiation by specific antibody against the sequences from intron 12. Immunofluorescence staining indicated that the HD3α isoform co-localized with CD31-positive or α-smooth muscle actin-positive cells at different developmental stages of mouse embryos. Overexpression of HD3α reprogrammed human aortic endothelial cells into mesenchymal cells featuring an endothelial-to-mesenchymal transition (EndMT) phenotype. HD3α directly interacts with HDAC3 and Akt1 and selectively activates transforming growth factor β2 (TGFβ2) secretion and cleavage. TGFβ2 functioned as an autocrine and/or paracrine EndMT factor. The HD3α-induced EndMT was both PI3K/Akt- and TGFβ2-dependent. This study provides the first evidence of the role of HDAC3 splicing in the maintenance of endothelial integrity.