Type 1 diabetes (T1D) is an autoimmune disease characterized by insulitis and islet β-cell loss. Thus, an effective therapy may require β-cell restoration and immune suppression. Currently, there is no treatment that can achieve both goals efficiently. We report here that GABA exerts antidiabetic effects by acting on both the islet β-cells and immune system. Unlike in adult brain or islet α-cells in which GABA exerts hyperpolarizing effects, in islet β-cells, GABA produces membrane depolarization and Ca 2+ influx, leading to the activation of PI3-K/Akt–dependent growth and survival pathways. This provides a potential mechanism underlying our in vivo findings that GABA therapy preserves β-cell mass and prevents the development of T1D. Remarkably, in severely diabetic mice, GABA restores β-cell mass and reverses the disease. Furthermore, GABA suppresses insulitis and systemic inflammatory cytokine production. The β-cell regenerative and immunoinhibitory effects of GABA provide insights into the role of GABA in regulating islet cell function and glucose homeostasis, which may find clinical application.
Transforming growth factor beta (TGF-beta) is a highly pleiotropic cytokine that plays an important role in wound healing, angiogenesis, immunoregulation and cancer. The cells of the immune system produce the TGF-beta1 isoform, which exerts powerful anti-inflammatory functions, and is a master regulator of the immune response. However, this is context dependent, because TGF-beta can contribute to the differentiation of both regulatory (suppressive) T cells (Tr cells) and inflammatory Th17 cells. While TGF-beta might be underproduced in some autoimmune diseases, it is overproduced in many pathological conditions. This includes pulmonary fibrosis, glomerulosclerosis, renal interstitial fibrosis, cirrhosis, Crohn's disease, cardiomyopathy, scleroderma and chronic graft-vs-host disease. In neoplastic disease, TGF-beta suppresses the progression of early lesions, but later this effect is lost and cancer cells produce TGF-beta, which then promotes metastasis. This cytokine also contributes to the formation of the tumor stroma, angiogenesis and immunosuppression. In view of this, several approaches are being studied to inhibit TGF-beta activity, including neutralizing antibodies, soluble receptors, receptor kinase antagonist drugs, antisense reagents and a number of less specific drugs such as angiotensin II antagonists and tranilast. It might be assumed that TGF-beta blockade would result in severe inflammatory disease, but this has not been the case, presumably because the neutralization is only partial. In contrast, the systemic administration of TGF-beta for therapeutic purposes is limited by toxicity and safety concerns, but local administration appears feasible, especially to promote wound healing. Immunotherapy or vaccination stimulating TGF-beta production and/or Tr differentiation might be applied to the treatment of autoimmune diseases. The benefits of new therapies targeting TGF-beta are under intense investigation.
The neuropilins (Nrps) are multifunctional proteins involved in development, immunity and cancer. Neuropilin-1 (Nrp1), or its homologue neuropilin-2 (Nrp2), are coreceptors that enhance responses to several growth factors (GFs) and other mediators. Nrps are coreceptors for the class 3 semaphorins (SEMA3), involved in axonal guidance, and several members of the vascular endothelial growth factor (VEGF) family. However, recent findings reveal they have a much broader spectrum of activity. They bind transforming growth factor β1 (TGF-β1) and its receptors, hepatocyte growth factor (HGF) and its receptor (cMet), platelet derived growth factor (PDGF) and its receptors, fibroblast growth factors (FGFs), and integrins. Nrps also promote Hedgehog signaling. These ligands and pathways are all relevant to angiogenesis and wound healing. In the immune system, the Nrps are expressed primarily by dendritic cells (DCs) and regulatory T cells (Tregs), and exert mainly inhibitory effects. In cancer, Nrps have been linked to a poor prognosis, which is consistent with their numerous interactions with ligands and receptors that promote tumor progression. We hypothesize that Nrps boost responses by capturing ligands, regulating GF receptor expression, endocytosis and recycling, and possibly also by signaling independently. Importantly, they promote epithelial-mesenchymal transition (EMT), and the survival of cancer stem cells. The recent finding that Nrps bind and internalize cell-penetrating peptides (CPPs) with arginine/lysine-rich C-terminal motifs (C-end rule; e.g., RXXR) is of interest. These CPPs can be coupled to large drugs for cancer therapy. Almost all studies have been preclinical, but findings suggest Nrps are excellent targets for anti-cancer drug development.
Neuropilin-1 (Nrp1) is a multifunctional protein, identified principally as a receptor for the class 3 semaphorins and members of the vascular endothelial growth factor (VEGF) family, but it is capable of other interactions. It is a marker of regulatory T cells (Tr), which often carry Nrp1 and latency-associated peptide (LAP)-TGF-β1 (the latent form). The signaling TGF-β1 receptors bind only active TGF-β1, and we hypothesized that Nrp1 binds the latent form. Indeed, we found that Nrp1 is a high-affinity receptor for latent and active TGF-β1. Free LAP, LAP-TGF-β1, and active TGF-β1 all competed with VEGF165 for binding to Nrp1. LAP has a basic, arginine-rich C-terminal motif similar to VEGF and peptides that bind to the b1 domain of Nrp1. A C-terminal LAP peptide (QSSRHRR) bound to Nrp1 and inhibited the binding of VEGF and LAP-TGF-β1. We also analyzed the effects of Nrp1/LAP-TGF-β1 coexpression on T cell function. Compared with Nrp1– cells, sorted Nrp1+ T cells had a much greater capacity to capture LAP-TGF-β1. Sorted Nrp1– T cells captured soluble Nrp1-Fc, and this increased their ability to capture LAP-TGF-β1. Conventional CD4+CD25–Nrp1– T cells coated with Nrp1-Fc/LAP-TGF-β1 acquired strong Tr activity. Moreover, LAP-TGF-β was activated by Nrp1-Fc and also by a peptide of the b2 domain of Nrp1 (RKFK; similar to a thrombospondin-1 peptide). Breast cancer cells, which express Nrp1, also captured and activated LAP-TGF-β1 in a Nrp1-dependent manner. Thus, Nrp1 is a receptor for TGF-β1, activates its latent form, and is relevant to Tr activity and tumor biology.
γ-Aminobutyric acid (GABA) exerts protective and regenerative effects on mouse islet β-cells. However, in humans it is unknown whether it can increase β-cell mass and improve glucose homeostasis. To address this question, we transplanted a suboptimal mass of human islets into immunodeficient NOD-scid-γ mice with streptozotocin-induced diabetes. GABA treatment increased grafted β-cell proliferation, while decreasing apoptosis, leading to enhanced β-cell mass. This was associated with increased circulating human insulin and reduced glucagon levels. Importantly, GABA administration lowered blood glucose levels and improved glucose excursion rates. We investigated GABA receptor expression and signaling mechanisms. In human islets, GABA activated a calcium-dependent signaling pathway through both GABA A receptor and GABA B receptor. This activated the phosphatidylinositol 3-kinase–Akt and CREB–IRS-2 signaling pathways that convey GABA signals responsible for β-cell proliferation and survival. Our findings suggest that GABA regulates human β-cell mass and may be beneficial for the treatment of diabetes or improvement of islet transplantation.
Neuropilin (Nrp)-1 and Nrp-2 are multifunctional proteins frequently expressed by cancer cells and contribute to tumor progression by mechanisms that are not well understood. They are co-receptors for vascular endothelial growth factor and class 3 semaphorins, but recently we found that Nrp1 also binds latent and active transforming growth factor (TGF)-β1, and activates the latent form latency-associated peptide (LAP)-TGF-β1. Here, we report that Nrp1 has affinity for TGF-β receptors TβRI and TβRII, the signaling TGF-β receptors, as well as TβRIII (betaglycan), as determined in binding assays, pull down assays and confocal microscopy. Nrp1 had a higher affinity for TβRI than TβRII and could form a complex with these receptors. In breast cancer cells, Nrp1 and TβRI cointernalized in the presence of TGF-β1. Nrp1 acted as a TGF-β co-receptor by augmenting canonical Smad2/3 signaling. Importantly, Nrp-positive cancer cells, unlike negative cells, were able to activate latent TGF-β1 and respond. We examined two other membrane proteins that bind LAP-TGF-β, i.e. an RGD-binding integrin (αvβ3) and Glycoprotein A repetitions predominant (CLRRC32). RGD-binding integrins are frequently expressed by cancer cells, and glycoprotein A repetitions predominant is expressed by activated regulatory T cells that appear linked to poor tumor immunity. In vitro, these receptors did not activate LAP-TGF-β1, but subsequent addition of Nrp1 activated the cytokine. Thus, Nrp1 might collaborate with other latent TGF-β receptors in TGF-β capture and activation. We also show that Nrp2 has activities similar to Nrp1. We conclude that Nrp1 is a co-receptor for TGF-β1 and augments responses to latent and active TGF-β. Since TGF-β promotes metastasis this is highly relevant to cancer biology.
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