CDK12-mediated transcriptional regulation of noncanonical NF-κB components is essential for signaling.

Members of the family of nuclear factor κB (NF-κB) transcription factors are critical for multiple cellular processes, including regulating innate and adaptive immune responses, cell proliferation, and cell survival. Canonical NF-κB complexes are retained in the cytoplasm by the inhibitory protein IκBα, whereas noncanonical NF-κB complexes are retained by p100. Although activation of canonical NF-κB signaling through the IκBα kinase complex is well studied, few regulators of the NF-κB-inducing kinase (NIK)-dependent processing of noncanonical p100 to p52 and the subsequent nuclear translocation of p52 have been identified. We discovered a role for cyclin-dependent kinase 12 (CDK12) in transcriptionally regulating the noncanonical NF-κB pathway. High-content phenotypic screening identified the compound 919278 as a specific inhibitor of the lymphotoxin ß receptor (LTßR), and tumor necrosis factor (TNF) receptor superfamily member 12A (FN14)-dependent nuclear translocation of p52, but not of the TNF-α receptor-mediated nuclear translocation of p65. Chemoproteomics identified CDK12 as the target of 919278. CDK12 inhibition by 919278, the CDK inhibitor THZ1, or siRNA-mediated knockdown resulted in similar global transcriptional changes and prevented the LTßR- and FN14-dependent expression of MAP3K14 (which encodes NIK) as well as NIK accumulation by reducing phosphorylation of the carboxyl-terminal domain of RNA polymerase II. By coupling a phenotypic screen with chemoproteomics, we identified a pathway for the activation of the noncanonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.

Glucose-regulated protein 78 autoantibody associates with blood-brain barrier disruption in neuromyelitis optica.

Neuromyelitis optica (NMO) is an inflammatory disorder mediated by antibodies to aquaporin-4 (AQP4) with prominent blood-brain barrier (BBB) breakdown in the acute phase of the disease. Anti-AQP4 antibodies are produced mainly in the periphery, yet they target the astrocyte perivascular end feet behind the BBB. We reasoned that an endothelial cell-targeted autoantibody might promote BBB transit of AQP4 antibodies and facilitate NMO attacks. Using monoclonal recombinant antibodies (rAbs) from patients with NMO, we identified two that strongly bound to the brain microvascular endothelial cells (BMECs). Exposure of BMECs to these rAbs resulted in nuclear translocation of nuclear factor κB p65, decreased claudin-5 protein expression, and enhanced transit of macromolecules. Unbiased membrane proteomics identified glucose-regulated protein 78 (GRP78) as the rAb target. Using immobilized GRP78 to deplete GRP78 antibodies from pooled total immunoglobulin G (IgG) of 50 NMO patients (NMO-IgG) reduced the biological effect of NMO-IgG on BMECs. GRP78 was expressed on the surface of murine BMECs in vivo, and repeated administration of a GRP78-specific rAb caused extravasation of serum albumin, IgG, and fibrinogen into mouse brains. Our results identify GRP78 antibodies as a potential component of NMO pathogenesis and GRP78 as a candidate target for promoting central nervous system transit of therapeutic antibodies.

IGFBP-5 Metabolism Is Disrupted in the Rat Medial Meniscal Tear Model of Osteoarthritis.

Insulin-like growth factor binding protein 5 (IGFBP-5) has been proposed to promote cartilage anabolism through insulin-like growth factor (IGF-1) signaling. A proteolytic activity towards IGFBP-5 has been detected in synovial fluids from human osteoarthritic (OA) joints. The purpose of this study was to determine if protease activity towards IGFBP-5 is present in the rat medial meniscal tear (MMT) model of OA and whether inhibition of this activity would alter disease progression. Sprague-Dawley rats were subject to MMT surgery. Synovial fluid lavages were assessed for the presence of IGFBP-5 proteolytic activity. Treatment animals received intra-articular injections of vehicle or protease inhibitor peptide PB-145. Cartilage lesions were monitored by India ink staining followed by macroscopic measurement of lesion width and depth. The MMT surgery induced a proteolytic activity towards IGFPB-5 that was detectable in joint fluid. This activity was stimulated by calcium and was sensitive to serine protease inhibitors as well as peptide PB-145. Significantly, intra-articular administration of PB-145 after surgery protected cartilage from lesion development. PB-145 treatment also resulted in an increase in cartilage turnover as evidenced by increases in serum levels of procollagen type II C-propeptide (CPII) as well as synovial fluid lavage levels of collagen type II neoepitope (TIINE). IGFBP-5 metabolism is disrupted in the rat MMT model of OA, potentially contributing to cartilage degradation. Inhibition of IGFBP-5 proteolysis protected cartilage from lesion development and enhanced cartilage turnover. These data are consistent with IGFBP-5 playing a positive role in anabolic IGF signaling in cartilage.