The ability of immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptors to inhibit, rather than activate, signaling by other receptors is a regulatory mechanism of immune homeostasis. However, it remains unclear how inhibitory ITAM (ITAMi) receptor signaling and Src homology 2 (SH2) domain-containing phosphatase-1 (SHP-1), which is recruited to ITAMs, target multiple heterologous activating responses without coaggregating with the associated activating receptors. We found that ITAMi signaling triggered by the binding of monomeric ligands to the type I immunoglobulin A (IgA) Fc receptor (FcαRI) induced its dynamic cosegregation with heterologous activating receptors, signaling effectors, and the inhibitory phosphatase SHP-1 into polarized intracellular clusters that we call "inhibisomes." Formation of inhibisomes was preceded by the recruitment of FcαRI and SHP-1 into lipid rafts. Cosegregation required the depolymerization of actin, which depended on SHP-1, and inhibisome formation was abolished by knockdown of SHP-1 and by actin-depolymerizing drugs. Thus, SHP-1- and actin depolymerization-dependent spatiotemporal compartmentalization of ITAMi-containing receptors into lipid rafts, regions associated with intracellular signaling, represents a key event in the integration of ITAMi-mediated inhibitory signals.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and has significant morbidity and mortality as 20–40% of patients progress to end-stage renal disease (ESRD) within 20 years after disease onset. We aimed to gain insight into the molecular mechanisms involved in IgAN progression. A systematic evaluation of renal biopsy specimens from IgAN patients revealed that the MAPK/ERK signaling pathway was activated in mesangial areas of patients presenting with >1 g/day proteinuria and elevated blood pressure, but was absent in biopsy specimens from IgAN patients with modest proteinuria (<1 g/day). ERK activation was not associated with elevated serum levels of galactose (Gal)-deficient IgA1 or IgG specific for Gal-deficient IgA1. In in vitro studies with human mesangial cells, ERK activation controlled pro-inflammatory cytokine secretion and was induced by patients’ large-molecular-mass IgA1-containing circulating immune complexes. Moreover, we show that IgA1-dependent MAPK/ERK activation required renin-angiotensin system (RAS) activity. Finally, RAS blockers were more efficient in reducing proteinuria in IgAN patients exhibiting substantial mesangial activation of MAPK/ERK. Together, these results suggest that MAPK/ERK activation alters the mesangial cell-podocyte cross-talk, leading to renal dysfunction in IgAN. Assessment of MAPK/ERK activation status in diagnostic renal biopsies could therefore serve as a biomarker to predict the efficacy of RAS blockers in IgAN.

Anemia because of insufficient production of and/or response to erythropoietin (Epo) is a major complication of chronic kidney disease and cancer. The mechanisms modulating the sensitivity of erythroblasts to Epo remain poorly understood. We show that, when cultured with Epo at suboptimal concentrations, the growth and clonogenic potential of erythroblasts was rescued by transferrin receptor 1 (TfR1)-bound polymeric IgA1 (pIgA1). Under homeostatic conditions, erythroblast numbers were increased in mice expressing human IgA1 compared to control mice. Hypoxic stress of these mice led to increased amounts of pIgA1 and erythroblast expansion. Expression of human IgA1 or treatment of wild-type mice with the TfR1 ligands pIgA1 or iron-loaded transferrin (Fe-Tf) accelerated recovery from acute anemia. TfR1 engagement by either pIgA1 or Fe-Tf increased cell sensitivity to Epo by inducing activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. These cellular responses were mediated through the TfR1-internalization motif, YXXΦ. Our results show that pIgA1 and TfR1 are positive regulators of erythropoiesis in both physiological and pathological situations. Targeting this pathway may provide alternate approaches to the treatment of ineffective erythropoiesis and anemia.

Gene amplification drives oncogenesis in a broad spectrum of cancers. A number of drugs have been developed to inhibit the protein products of amplified driver genes, but their clinical efficacy is often hampered by drug resistance. Here, we introduce a therapeutic strategy for targeting cancer-associated gene amplifications by activating the DNA damage response with triplex-forming oligonucleotides (TFOs), which drives induction of apoptosis in tumors, whereas cells without amplifications process lower levels of DNA damage. Focusing on cancers driven by HER2-amplification, we find that TFOs targeting HER2 induce copy number-dependent DNA double strand breaks and activate p53-independent apoptosis in HER2-positive cancer cells and human tumor xenografts via a mechanism that is independent of HER2 cellular function. This strategy has demonstrated in vivo efficacy comparable with current precision medicines and provided a feasible alternative to combat drug resistance in HER-positive breast and ovarian cancer models. These findings offer a general strategy for targeting tumors with amplified genomic loci.

Structural alterations in DNA can serve as natural impediments to replication fork stability and progression, resulting in DNA damage and genomic instability. Naturally occurring polypurine mirror repeat sequences in the human genome can create endogenous triplex structures evoking a robust DNA damage response. Failures to recognize or adequately process these genomic lesions can result in loss of genomic integrity. Nucleotide excision repair (NER) proteins have been found to play a prominent role in the recognition and repair of triplex structures. We demonstrate using triplex-forming oligonucleotides that chromosomal triplexes perturb DNA replication fork progression, eventually resulting in fork collapse and the induction of double strand breaks (DSBs). We find that cells deficient in the NER damage recognition proteins, XPA and XPC, accumulate more DSBs in response to chromosomal triplex formation than NER-proficient cells. Furthermore, we demonstrate that XPC-deficient cells are particularly prone to replication-associated DSBs in the presence of triplexes. In the absence of XPA or XPC, deleterious consequences of triplex-induced genomic instability may be averted by activating apoptosis via dual phosphorylation of the H2AX protein. Our results reveal that damage recognition by XPC and XPA is critical to maintaining replication fork integrity and preventing replication fork collapse in the presence of triplex structures.

DNA sequences capable of forming triplexes are prevalent in the human genome and have been found to be intrinsically mutagenic. Consequently, a balance between DNA repair and apoptosis is critical to counteract their effect on genomic integrity. Using triplex-forming oligonucleotides to synthetically create altered helical distortions, we have determined that pro-apoptotic pathways are activated by the formation of triplex structures. Moreover, the TFIIH factor, XPD, occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. Here, we show that triplexes are capable of inducing XPD-independent double strand breaks, which result in the formation of γH2AX foci. XPD was subsequently recruited to the triplex-induced double strand breaks and co-localized with γH2AX at the damage site. Furthermore, phosphorylation of H2AX tyrosine 142 was found to stimulate the signaling pathway of XPD-dependent apoptosis. We suggest that this mechanism may play an active role in minimizing genomic instability induced by naturally occurring noncanonical structures, perhaps protecting against cancer initiation.

IgA nephropathy (IgAN) is characterized by IgA immune complex-mediated mesangial cell proliferation. We have previously identified the transferrin receptor (TfR) as an IgA1 receptor and found that, in kidney biopsies of patients with IgAN, TfR is overexpressed and co-localized with IgA1 mesangial deposits. We also showed that IgA1 binding to TfR was strikingly increased when IgA1 was hypogalactosylated and of high molecular weight, both features found in IgA from IgAN patients. More recently, we showed that purified polymeric IgA1 (pIgA1) is a major inducer of TfR expression (3-fold increase) in quiescent human mesangial cells (HMC). In addition, sera from IgAN patients upregulate TfR expression in cultured HMC in an IgA-dependent manner. IgA1-induced HMC proliferation is dependent on TfR engagement and can be inhibited by both TfR1 and TfR2 ectodomains as well as by the anti-TfR mAb A24. Finally, activation of mesangial cells through pIgA1 binding to TfR induced secretion of IL-6 and TGF-beta from the cells, that could be involved, respectively, in the inflammatory and pro-fibrogenic events observed in IgAN. We propose that deposited pIgA1 or IgA immune complexes could initiate an auto-amplification process involving hyper-expression of TfR allowing increased IgA1 mesangial deposition. Altogether, these data unveil a functional cooperation between pIgA1 and TfR for IgA1 deposition and HMC proliferation, features which are commonly implicated in the chronic mesangial injuries observed in IgAN.

No abstract