An antibody that inhibits TGF-ß1 release from latent extracellular matrix complexes attenuates the progression of renal fibrosis.

Inhibitors of the transforming growth factor-ß (TGF-ß) pathway are potentially promising antifibrotic therapies, but nonselective simultaneous inhibition of all three TGF-ß homologs has safety liabilities. TGF-ß1 is noncovalently bound to a latency-associated peptide that is, in turn, covalently bound to different presenting molecules within large latent complexes. The latent TGF-ß-binding proteins (LTBPs) present TGF-ß1 in the extracellular matrix, and TGF-ß1 is presented on immune cells by two transmembrane proteins, glycoprotein A repetitions predominant (GARP) and leucine-rich repeat protein 33 (LRRC33). Here, we describe LTBP-49247, an antibody that selectively bound to and inhibited the activation of TGF-ß1 presented by LTBPs but did not bind to TGF-ß1 presented by GARP or LRRC33. Structural studies demonstrated that LTBP-49247 recognized an epitope on LTBP-presented TGF-ß1 that is not accessible on GARP- or LRRC33-presented TGF-ß1, explaining the antibody's selectivity for LTBP-complexed TGF-ß1. In two rodent models of kidney fibrosis of different etiologies, LTBP-49247 attenuated fibrotic progression, indicating the central role of LTBP-presented TGF-ß1 in renal fibrosis. In mice, LTBP-49247 did not have the toxic effects associated with less selective TGF-ß inhibitors. These results establish the feasibility of selectively targeting LTBP-bound TGF-ß1 as an approach for treating fibrosis.

Click Chemistry Selectively Activates an Auristatin Protodrug with either Intratumoral or Systemic Tumor-Targeting Agents.

The Click Activated Protodrugs Against Cancer (CAPAC) platform enables the activation of powerful cancer drugs at tumors. CAPAC utilizes a click chemistry reaction between tetrazine and trans-cyclooctene. The reaction between activator, linked to a tumor-targeting agent, and protodrug leads to the targeted activation of the drug. Here, tumor targeting is achieved by intratumoral injection of a tetrazine-modified hyaluronate (SQL70) or by infusion of a tetrazine-modified HER2-targeting antigen-binding fragment (SQT01). Monomethyl auristatin E (a cytotoxin hindered in its clinical use by severe toxicity) was modified with a trans-cyclooctene to form the protodrug SQP22, which reduced its cytotoxicity in vitro and in vivo. Treatment of SQP22 paired with SQL70 demonstrated antitumor effects in Karpas 299 and RENCA murine tumor models, establishing the requirement of click chemistry for protodrug activation. SQP22 paired with SQT01 induced antitumor effects in the HER2-positive NCI-N87 xenograft model, showing that tumor-targeted activation could be accomplished via systemic dosing. Observed toxicities were limited, with transient myelosuppression and moderate body weight loss detected. This study highlights the capabilities of the CAPAC platform by demonstrating the activity of SQP22 with two differentiated targeting approaches and underscores the power of click chemistry to precisely control the activation of drugs at tumors.

Mechanism Governing Human Kappa-Opioid Receptor Expression under Desferrioxamine-Induced Hypoxic Mimic Condition in Neuronal NMB Cells.

Cellular adaptation to hypoxia is a protective mechanism for neurons and relevant to cancer. Treatment with desferrioxamine (DFO) to induce hypoxia reduced the viability of human neuronal NMB cells. Surviving/attached cells exhibited profound increases of expression of the human kappa-opioid receptor (hKOR) and hypoxia inducible factor-1α (HIF-1α). The functional relationship between hKOR and HIF-1α was investigated using RT-PCR, Western blot, luciferase reporter, mutagenesis, siRNA and receptor-ligand binding assays. In surviving neurons, DFO increased HIF-1α expression and its amount in the nucleus. DFO also dramatically increased hKOR expression. Two (designated as HIFC and D) out of four potential HIF response elements of the hKOR gene (HIFA-D) synergistically mediated the DFO response. Mutation of both elements completely abolished the DFO-induced effect. The CD11 plasmid (containing HIFC and D with an 11 bp spacing) produced greater augmentation than that of the CD17 plasmid (HIFC and D with a 17 bp-spacing), suggesting that a proper topological interaction of these elements synergistically enhanced the promoter activity. HIF-1α siRNA knocked down the increase of endogenous HIF-1α messages and diminished the DFO-induced increase of hKOR expression. Increased hKOR expression resulted in the up-regulation of hKOR protein. In conclusion, the adaptation of neuronal hKOR under hypoxia was governed by HIF-1, revealing a new mechanism of hKOR regulation.

TGF-ß regulates phosphorylation and stabilization of Sox9 protein in chondrocytes through p38 and Smad dependent mechanisms.

Members of the TGF-ß superfamily are important regulators of chondrocyte function. Sox9, a key transcriptional regulator of chondrogenesis, is required for TGF-ß-mediated regulation of specific cartilage genes. TGF-ß can signal through a canonical, Smad-mediated pathway or non-conical pathways, including p38. Here we show that both pathways are activated in chondrocytes after treatment with TGF-ß and that TGF-ß stabilizes Sox9 protein and increases phosphorylation of Sox9. Mutagenesis of potential serine phosphorylation sites on Sox9 was used to demonstrate that serine 211 is required to maintain normal basal levels of Sox9 as well as mediate increased Sox9 levels in response to TGF-ß. The serine 211 site is in a motif that is targeted by p38 kinase. We used siRNA and pharmacological agents to show that p38 and Smad3 independently regulate the phosphorylation and stability of Sox9. Previously, we demonstrated that Papss2 is a downstream transcriptional target of Sox9 and TGF-ß. Here we show that p38 is required for TGF-ß-mediated regulation of Papss2 mRNA. Together the results suggest a new mechanism for TGF-ß-mediated gene regulation in chondrocytes via p38 and phosphorylation and stabilization of Sox9. Understanding how TGF-ß regulates Sox9 may lead to identification of therapeutic targets for OA.

Cystic fibrosis transmembrane conductance regulator activation by roflumilast contributes to therapeutic benefit in chronic bronchitis.

Cigarette smoking causes acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction and is associated with delayed mucociliary clearance and chronic bronchitis. Roflumilast is a clinically approved phosphodiesterase 4 inhibitor that improves lung function in patients with chronic bronchitis. We hypothesized that its therapeutic benefit was related in part to activation of CFTR. Primary human bronchial epithelial (HBE) cells, Calu-3, and T84 monolayers were exposed to whole cigarette smoke (WCS) or air with or without roflumilast treatment. CFTR-dependent ion transport was measured in modified Ussing chambers. Airway surface liquid (ASL) was determined by confocal microscopy. Intestinal fluid secretion of ligated murine intestine was monitored ex vivo. Roflumilast activated CFTR-dependent anion transport in normal HBE cells with a half maximal effective concentration of 2.9 nM. Roflumilast partially restored CFTR activity in WCS-exposed HBE cells (5.3 ± 1.1 µA/cm(2) vs. 1.2 ± 0.2 µA/cm(2) [control]; P < 0.05) and was additive with ivacaftor, a specific CFTR potentiator approved for the treatment of CF. Roflumilast improved the depleted ASL depth of HBE monolayers exposed to WCS (9.0 ± 3.1 µm vs. 5.6 ± 2.0 µm [control]; P < 0.05), achieving 79% of that observed in air controls. CFTR activation by roflumilast also induced CFTR-dependent fluid secretion in murine intestine, increasing the wet:dry ratio and the diameter of ligated murine segments. Roflumilast activates CFTR-mediated anion transport in airway and intestinal epithelia via a cyclic adenosine monophosphate-dependent pathway and partially reverses the deleterious effects of WCS, resulting in augmented ASL depth. Roflumilast may benefit patients with chronic obstructive pulmonary disease with chronic bronchitis by activating CFTR, which may also underlie noninfectious diarrhea caused by roflumilast.

Inactivation of Tgfbr2 in Osterix-Cre expressing dental mesenchyme disrupts molar root formation.

It has been difficult to examine the role of TGF-ß in post-natal tooth development due to perinatal lethality in many of the signaling deficient mouse models. To address the role of Tgfbr2 in postnatal tooth development, we generated a mouse in which Tgfbr2 was deleted in odontoblast- and bone-producing mesenchyme. Osx-Cre;Tgfbr2(fl/fl) mice were generated (Tgfbr2(cko)) and post-natal tooth development was compared in Tgfbr2(cko) and control littermates. X-ray and µCT analysis showed that in Tgfbr2(cko) mice radicular dentin matrix density was reduced in the molars. Molar shape was abnormal and molar eruption was delayed in the mutant mice. Most significantly, defects in root formation, including failure of the root to elongate, were observed by postnatal day 10. Immunostaining for Keratin-14 (K14) was used to delineate Hertwig's epithelial root sheath (HERS). The results showed a delay in elongation and disorganization of the HERS in Tgfbr2(cko) mice. In addition, the HERS was maintained and the break up into epithelial rests was attenuated suggesting that Tgfbr2 acts on dental mesenchyme to indirectly regulate the formation and maintenance of the HERS. Altered odontoblast organization and reduced Dspp expression indicated that odontoblast differentiation was disrupted in the mutant mice likely contributing to the defect in root formation. Nevertheless, expression of Nfic, a key mesenchymal regulator of root development, was similar in Tgfbr2(cko) mice and controls. The number of osteoclasts in the bone surrounding the tooth was reduced and osteoblast differentiation was disrupted likely contributing to both root and eruption defects. We conclude that Tgfbr2 in dental mesenchyme and bone is required for tooth development particularly root formation.