CX3CL1--a macrophage chemoattractant induced by a single bout of exercise in human skeletal muscle.

Monocytes/macrophages (MOs/MΦs) are suggested to be crucial for skeletal muscle repair and remodeling. This has been attributed to their proangiogenic potential, secretion of growth factors, and clearance of tissue debris. Skeletal muscle injury increases the number of MΦs in the tissue, and their importance for muscle regeneration has been supported by studies demonstrating that depletion of MOs/MΦs greatly impairs repair after muscle injury. Whether noninjurious exercise leads to induced expression of chemoattractants for MOs/MΦs is poorly investigated. To this end, we analyzed the expression of CX3CL1 (fractalkine), CCL2 (MCP-1), and CCL22 (MDC) in human skeletal muscle after a bout of exercise, all of which are established MO/MΦ chemotactic factors that are expressed by human myoblasts. Muscle biopsies from the musculus vastus lateralis were obtained up to 24 h after 1 h of cycle exercise in healthy individuals and in age-matched nonexercised controls. CX3CL1 increased at both the mRNA and protein level in human skeletal muscle after one bout of exercise. It was not possible to distinguish changes in CCL2 or CCL22 mRNA levels between biopsy vs. exercise effects, and the expression of CCL22 was very low. CX3CL1 mainly localized to the skeletal muscle endothelium, and it increased in human umbilical vein endothelial cells stimulated with tissue fluid from exercised muscle. CX3CL1 increased the expression of proinflammatory and proangiogenic factors in THP-1 monocytes (a human acute monocytic leukemia cell line) and in human primary myoblasts and myotubes. Altogether, this suggests that CX3CL1 participates in cross-talk mechanisms between endothelium and other muscle tissue cells and may promote a shift in the microenvironment toward a more regenerative milieu.

High levels of WNT-5A in human glioma correlate with increased presence of tumor-associated microglia/monocytes.

Malignant gliomas are among the most severe types of cancer, and the most common primary brain tumors. Treatment options are limited and the prognosis is poor. WNT-5A, a member of the WNT family of lipoglycoproteins, plays a role in oncogenesis and tumor progression in various cancers, whereas the role of WNT-5A in glioma remains obscure. Based on the role of WNT-5A as an oncogene, its potential to regulate microglia cells and the glioma-promoting capacities of microglia cells, we hypothesize that WNT-5A has a role in regulation of immune functions in glioma. We investigated WNT-5A expression by in silico analysis of the cancer genome atlas (TCGA) transcript profiling of human glioblastoma samples and immunohistochemistry experiments of human glioma tissue microarrays (TMA). Our results reveal higher WNT-5A protein levels and mRNA expression in a subgroup of gliomas (WNT-5A(high)) compared to non-malignant control brain tissue. Furthermore, we show a significant correlation between WNT-5A in the tumor and presence of major histocompatibility complex Class II-positive microglia/monocytes. Our data pinpoint a positive correlation between WNT-5A and a proinflammatory signature in glioma. We identify increased presence of microglia/monocytes as an important aspect in the inflammatory transformation suggesting a novel role for WNT-5A in human glioma.

Systematic mapping of WNT-FZD protein interactions reveals functional selectivity by distinct WNT-FZD pairs.

The seven-transmembrane-spanning receptors of the FZD1-10 class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD2, FZD4, or FZD5, we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-ß-catenin pathway through FZD2/4/5 as measured by phosphorylation of LRP6 and ß-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain interactions complemented by analysis of WNT-FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs.

Sequential activation and inactivation of Dishevelled in the Wnt/beta-catenin pathway by casein kinases.

Dishevelled (Dvl) is a key component in the Wnt/ß-catenin signaling pathway. Dvl can multimerize to form dynamic protein aggregates, which are required for the activation of downstream signaling. Upon pathway activation by Wnts, Dvl becomes phosphorylated to yield phosphorylated and shifted (PS) Dvl. Both activation of Dvl in Wnt/ß-catenin signaling and Wnt-induced PS-Dvl formation are dependent on casein kinase 1 (CK1) δ/ε activity. However, the overexpression of CK1 was shown to dissolve Dvl aggregates, and endogenous PS-Dvl forms irrespective of whether or not the activating Wnt triggers the Wnt/ß-catenin pathway. Using a combination of gain-of-function, loss-of-function, and domain mapping approaches, we attempted to solve this discrepancy regarding the role of CK1ε in Dvl biology. We analyzed mutual interaction of CK1δ/ε and two other Dvl kinases, CK2 and PAR1, in the Wnt/ß-catenin pathway. We show that CK2 acts as a constitutive kinase whose activity is required for the further action of CK1ε. Furthermore, we demonstrate that the two consequences of CK1ε phosphorylation are separated both spatially and functionally; first, CK1ε-mediated induction of TCF/LEF-driven transcription (associated with dynamic recruitment of Axin1) is mediated via a PDZ-proline-rich region of Dvl. Second, CK1ε-mediated formation of PS-Dvl is mediated by the Dvl3 C terminus. Furthermore, we demonstrate with several methods that PS-Dvl has decreased ability to polymerize with other Dvls and could, thus, act as the inactive signaling intermediate. We propose a multistep and multikinase model for Dvl activation in the Wnt/ß-catenin pathway that uncovers a built-in de-activation mechanism that is triggered by activating phosphorylation of Dvl by CK1δ/ε.

WNT-5A stimulates the GDP/GTP exchange at pertussis toxin-sensitive heterotrimeric G proteins.

The lipoglycoproteins of the WNT family act on seven transmembrane-spanning Class Frizzled receptors. Here, we show that WNT-5A evokes a proliferative response in a mouse microglia-like cell line (N13), which is sensitive to pertussis toxin, thus implicating the involvement of heterotrimeric G proteins of the G(i/o) family. We continue to show that WNT-5A stimulation of N13 membranes and permeabilized cells evokes the exchange of GDP for GTP of pertussis toxin-sensitive G proteins employing [γ-(35)S]GTP assay and activity state-specific antibodies to GTP-bound G(i) proteins. Our functional analysis of the PTX-sensitivity of WNT-induced G protein activation and PCR analysis of G protein and FZD expression patterns suggest that WNT-5A stimulation leads to the activation of G(i2/3) proteins in N13 cells possibly mediated by FZD(5), the predominant FZD expressed. In summary, we provide for the first time molecular proof that WNT-5A stimulation results in the activation of heterotrimeric G(i2/3) proteins in mammalian cells with physiological protein stochiometry.