Targeting the molecular and cellular interactions of the bone marrow niche in immunologic disease.

Recent investigations have expanded our knowledge of the regulatory bone marrow (BM) niche, which is critical in maintaining and directing hematopoietic stem cell (HSC) self-renewal and differentiation. Osteoblasts, mesenchymal stem cells (MSCs), and CXCL12-abundant reticular (CAR) cells are niche components in close association with HSCs and have been more clearly defined in immune cell function and homeostasis. Importantly, cellular inhabitants of the BM niche signal through G protein-coupled surface receptors (GPCRs) for various appropriate immune functions. In this article, recent literature on BM niche inhabitants (HSCs, osteoblasts, MSCs, CAR cells) and their GPCR mechanistic interactions are reviewed for better understanding of the BM cells involved in immune development, immunologic disease, and current immune reconstitution therapies.

Aged G Protein-Coupled Receptor Kinase 3 (Grk3)-Deficient Mice Exhibit Enhanced Osteoclastogenesis and Develop Bone Lesions Analogous to Human Paget's Disease of Bone.

Paget's Disease of Bone (PDB) is a metabolic bone disease that is characterized by dysregulated osteoclast function leading to focal abnormalities of bone remodeling. It can lead to pain, fracture, and bone deformity. G protein-coupled receptor kinase 3 (GRK3) is an important negative regulator of G protein-coupled receptor (GPCR) signaling. GRK3 is known to regulate GPCR function in osteoblasts and preosteoblasts, but its regulatory function in osteoclasts is not well defined. Here, we report that Grk3 expression increases during osteoclast differentiation in both human and mouse primary cells and established cell lines. We also show that aged mice deficient in Grk3 develop bone lesions similar to those seen in human PDB and other Paget's Disease mouse models. We show that a deficiency in Grk3 expression enhances osteoclastogenesis in vitro and proliferation of hematopoietic osteoclast precursors in vivo but does not affect the osteoclast-mediated bone resorption function or cellular senescence pathway. Notably, we also observe decreased Grk3 expression in peripheral blood mononuclear cells of patients with PDB compared with age- and gender-matched healthy controls. Our data suggest that GRK3 has relevance to the regulation of osteoclast differentiation and that it may have relevance to the pathogenesis of PDB and other metabolic bone diseases associated with osteoclast activation.

G protein-coupled receptor kinase 3 modulates mesenchymal stem cell proliferation and differentiation through sphingosine-1-phosphate receptor regulation.

BACKGROUND: The bone marrow niche supports hematopoietic cell development through intimate contact with multipotent stromal mesenchymal stem cells; however, the intracellular signaling, function, and regulation of such supportive niche cells are still being defined. Our study was designed to understand how G protein receptor kinase 3 (GRK3) affects bone marrow mesenchymal stem cell function by examining primary cells from GRK3-deficient mice, which we have previously published to have a hypercellular bone marrow and leukocytosis through negative regulation of CXCL12/CXCR4 signaling. METHODS: Murine GRK3-deficient bone marrow mesenchymal stromal cells were harvested and cultured to differentiate into three lineages (adipocyte, chondrocyte, and osteoblast) to confirm multipotency and compared to wild type cells. Immunoblotting, modified-TANGO experiments, and flow cytometry were used to further examine the effects of GRK3 deficiency on bone marrow mesenchymal stromal cell receptor signaling. Microcomputed tomography was used to determine trabecular and cortical bone composition of GRK3-deficient mice and standard ELISA to quantitate CXCL12 production from cellular cultures. RESULTS: GRK3-deficient, bone marrow-derived mesenchymal stem cells exhibit enhanced and earlier osteogenic differentiation in vitro. The addition of a sphingosine kinase inhibitor abrogated the osteogenic proliferation and differentiation, suggesting that sphingosine-1-phosphate receptor signaling was a putative G protein-coupled receptor regulated by GRK3. Immunoblotting showed prolonged ERK1/2 signaling after stimulation with sphingosine-1-phosphate in GRK3-deficient cells, and modified-TANGO assays suggested the involvement of ß-arrestin-2 in sphingosine-1-phosphate receptor internalization. CONCLUSIONS: Our work suggests that GRK3 regulates sphingosine-1-phosphate receptor signaling on bone marrow mesenchymal stem cells by recruiting ß-arrestin to the occupied GPCR to promote internalization, and lack of such regulation affects mesenchymal stem cell functionality.

Chemerin-activated functions of CMKLR1 are regulated by G protein-coupled receptor kinase 6 (GRK6) and ß-arrestin 2 in inflammatory macrophages.

Chemerin receptor (CMKLR1) is a G protein-coupled receptor (GPCR) implicated in macrophage-mediated inflammation and in several forms of human arthritis. Analogous to other GPCR, CMKLR1 is likely regulated by G protein-coupled receptor kinase (GRK) phosphorylation of intracellular domains in an activation-dependent manner, which leads to recruitment and termination of intracellular signaling via desensitization and internalization of the receptor. The ubiquitously expressed GRK family members include GRK2, GRK3, GRK5, and GRK6, but it is unknown which GRK regulates CMKLR1 cellular and signaling functions. Our data show that activation of CMKLR1 by chemerin in primary macrophages leads to signaling and functional outcomes that are regulated by GRK6 and ß-arrestin 2. We show that arrestin recruitment to CMKLR1 following chemerin stimulation is enhanced with co-expression of GRK6. Further, internalization of endogenous CMKLR1, following the addition of chemerin, is decreased in inflammatory macrophages from GRK6- and ß-arrestin 2-deficient mice. These GRK6- and ß-arrestin 2-deficient macrophages display increased migration toward chemerin and altered AKT and Extracellular-signal Related Kinase (ERK) signaling. Our findings show that chemerin-activated CMKLR1 regulation in inflammatory macrophages is largely GRK6 and ß-arrestin mediated, which may impact innate immunity and have therapeutic implications in rheumatic disease.

The dysfunction of BP180/collagen XVII in keratinocytes promotes melanoma progression.

BP180, also termed collagen XVII, is a hemidesmosomal transmembrane glycoprotein expressed in basal keratinocytes, and functions as a cell-matrix adhesion molecule in the dermal-epidermal junction of the skin. Its function, other than cell-matrix adhesion, remains unclear. We generated a mouse strain with BP180 dysfunction (termed ∆NC16A), which develops spontaneous skin inflammation accompanied by an influx of myeloid derived suppressor cells (MDSCs). We used the B16 mouse melanoma model to demonstrate that BP180 dysfunction in either skin or basal keratinocytes promotes MDSC influx into skin and tumor progression. MDSC depletion reduced tumor progression in ∆NC16A mice, demonstrating a critical role for BP180 dysfunction-driven MDSCs in melanoma progression. This study provides the first direct evidence that BP180, a cell-cell matrix adhesion molecule, possesses antitumor function through modulating infiltration of MDSCs. Basal keratinocytes actively participate in skin microenvironment changes caused by BP180 dysfunction. ∆NC16A mice could be a new animal model to study the melanoma microenvironment.

G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis.

Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.