Urokinase Plasminogen Activator Inhibits HIV Virion Release from Macrophage-Differentiated Chronically Infected Cells via Activation of RhoA and PKCε

HIV replication in mononuclear phagocytes is a multi-step process regulated by viral and cellular proteins with the peculiar feature of virion budding and accumulation in intra-cytoplasmic vesicles. Interaction of urokinase-type plasminogen activator (uPA) with its cell surface receptor (uPAR) has been shown to favor virion accumulation in such sub-cellular compartment in primary monocyte-derived macrophages and chronically infected promonocytic U1 cells differentiated into macrophage-like cells by stimulation with phorbol myristate acetate (PMA). By adopting this latter model system, we have here investigated which intracellular signaling pathways were triggered by uPA/uPAR interaction leading the redirection of virion accumulation in intra-cytoplasmic vesicles.uPA induced activation of RhoA, PKCδ and PKCε in PMA-differentiated U1 cells. In the same conditions, RhoA, PKCδ and PKCε modulated uPA-induced cell adhesion and polarization, whereas only RhoA and PKCε were also responsible for the redirection of virions in intracellular vesicles. Distribution of G and F actin revealed that uPA reorganized the cytoskeleton in both adherent and polarized cells. The role of G and F actin isoforms was unveiled by the use of cytochalasin D, a cell-permeable fungal toxin that prevents F actin polymerization. Receptor-independent cytoskeleton remodeling by Cytochalasin D resulted in cell adhesion, polarization and intracellular accumulation of HIV virions similar to the effects gained with uPA.These findings illustrate the potential contribution of the uPA/uPAR system in the generation and/or maintenance of intra-cytoplasmic vesicles that actively accumulate virions, thus sustaining the presence of HIV reservoirs of macrophage origin. In addition, our observations also provide evidences that pathways controlling cytoskeleton remodeling and activation of PKCε bear relevance for the design of new antiviral strategies aimed at interfering with the partitioning of virion budding between intra-cytoplasmic vesicles and plasma membrane in infected human macrophages

Genome-wide protein QTL mapping identifies human plasma kallikrein as a post-translational regulator of serum uPAR levels

The soluble cleaved urokinase plasminogen activator receptor (scuPAR) is a circulating protein detected in multiple diseases, including various cancers, cardiovascular disease, and kidney disease, where elevated levels of scuPAR have been associated with worsening prognosis and increased disease aggressiveness. We aimed to identify novel genetic and biomolecular mechanisms regulating scuPAR levels. Elevated serum scuPAR levels were identified in asthma (n=514) and chronic obstructive pulmonary disease (COPD; n=219) cohorts when compared to controls (n=96). In these cohorts, a genome-wide association study of serum scuPAR levels identified a human plasma kallikrein gene (KLKB1) promoter polymorphism (rs4253238) associated with serum scuPAR levels in a control/asthma population (P=1.17×10−7), which was also observed in a COPD population (combined P=5.04×10−12). Using a fluorescent assay, we demonstrated that serum KLKB1 enzymatic activity was driven by rs4253238 and is inverse to scuPAR levels. Biochemical analysis identified that KLKB1 cleaves scuPAR and negates scuPAR's effects on primary human bronchial epithelial cells (HBECs) in vitro. Chymotrypsin was used as a proproteolytic control, while basal HBECs were used as a control to define scuPAR-driven effects. In summary, we reveal a novel post-translational regulatory mechanism for scuPAR using a hypothesis-free approach with implications for multiple human diseases

suPAR as a prognostic biomarker in sepsis

Sepsis is the clinical syndrome derived from the host response to an infection and severe sepsis is the leading cause of death in critically ill patients. Several biomarkers have been tested for use in diagnosis and prognostication in patients with sepsis. Soluble urokinase-type plasminogen activator receptor (suPAR) levels are increased in various infectious diseases, in the blood and also in other tissues. However, the diagnostic value of suPAR in sepsis has not been well defined, especially compared to other more established biomarkers, such as C-reactive protein (CRP) and procalcitonin (PCT). On the other hand, suPAR levels have been shown to predict outcome in various kinds of bacteremia and recent data suggest they may have predictive value, similar to that of severity scores, in critically ill patients. This narrative review provides a descriptive overview of the clinical value of this biomarker in the diagnosis, prognosis and therapeutic guidance of sepsis

C4.4A as a candidate marker in the diagnosis of colorectal cancer

C4.4A is a member of the Ly-6 family with restricted expression in non-transformed tissues. C4.4A expression in human cancer has rarely been evaluated. Thus, it became important to explore C4.4A protein expression in human tumour tissue to obtain an estimate on the frequency of expression and the correlation with tumour progression, the study focusing on colorectal cancer. The analysis of C4.4A in human tumour lines by western blot and immunoprecipitation using polyclonal rabbit antibodies that recognize different C4.4A epitopes revealed C4.4A oligomer and heavily glycosylated C4.4A isoform expression that, in some instances, inhibited antibody binding and interaction with the C4.4A ligand galectin-3. In addition, tumour cell lines released C4.4A by vesicle shedding and proteolytic cleavage. C4.4A was expressed in over 80% of primary colorectal cancer and liver metastasis with negligible expression in adjacent colonic mucosa, inflamed colonic tissue and liver. This compares well with EpCAM and CO-029 expression in over 90% of colorectal cancer. C4.4A expression was only observed in about 50% of pancreatic cancer and renal cell carcinoma. By de novo expression in colonic cancer tissue, we consider C4.4A as a candidate diagnostic marker in colorectal cancer, which possibly can be detected in body fluids

Host hindrance to HIV-1 replication in monocytes and macrophages

Monocytes and macrophages are targets of HIV-1 infection and play critical roles in multiple aspects of viral pathogenesis. HIV-1 can replicate in blood monocytes, although only a minor proportion of circulating monocytes harbor viral DNA. Resident macrophages in tissues can be infected and function as viral reservoirs. However, their susceptibility to infection, and their capacity to actively replicate the virus, varies greatly depending on the tissue localization and cytokine environment. The susceptibility of monocytes to HIV-1 infection in vitro depends on their differentiation status. Monocytes are refractory to infection and become permissive upon differentiation into macrophages. In addition, the capacity of monocyte-derived macrophages to sustain viral replication varies between individuals. Host determinants regulate HIV-1 replication in monocytes and macrophages, limiting several steps of the viral life-cycle, from viral entry to virus release. Some host factors responsible for HIV-1 restriction are shared with T lymphocytes, but several anti-viral mechanisms are specific to either monocytes or macrophages. Whilst a number of these mechanisms have been identified in monocytes or in monocyte-derived macrophages in vitro, some of them have also been implicated in the regulation of HIV-1 infection in vivo, in particular in the brain and the lung where macrophages are the main cell type infected by HIV-1. This review focuses on cellular factors that have been reported to interfere with HIV-1 infection in monocytes and macrophages, and examines the evidences supporting their role in vivo, highlighting unique aspects of HIV-1 restriction in these two cell types

Enhancement of cutaneous wound healing by Dsg2 augmentation of uPAR secretion

In addition to playing a role in adhesion, desmoglein 2 (Dsg2) is an important regulator of growth and survival signaling pathways, cell proliferation, migration and invasion, and oncogenesis. While low-level Dsg2 expression is observed in basal keratinocytes and is downregulated in non-healing venous ulcers, overexpression has been observed in both melanomas and non-melanoma malignancies. Here, we show that transgenic mice overexpressing Dsg2 in basal keratinocytes primed the activation of mitogenic pathways, but did not induce dramatic epidermal changes or susceptibility to chemical-induced tumor development. Interestingly, acceleration of full-thickness wound closure and increased wound-adjacent keratinocyte proliferation was observed in these mice. As epidermal cytokines and their receptors play critical roles in wound healing, Dsg2-induced secretome alterations were assessed with an antibody profiler array and revealed increased release and proteolytic processing of the urokinase-type plasminogen activator receptor (uPAR). Dsg2 induced uPAR expression in the skin of transgenic compared to wild-type mice. Wound healing further enhanced uPAR in both epidermis and dermis with concomitant increase in the pro-healing laminin-332, a major component of the basement membrane zone, in transgenic mice. This study demonstrates that Dsg2 induces epidermal activation of various signaling cascades and accelerates cutaneous wound healing, in part, through uPAR-related signaling cascades