Abstract
Calcification-related chronic inflammatory diseases are multifactorial pathological processes, involving a complex interplay between inflammation and calcification events in a positive feed-back loop driving disease progression. Gla-rich protein (GRP) is a vitamin K dependent protein (VKDP) shown to function as a calcification inhibitor in cardiovascular and articular tissues, and proposed as an anti-inflammatory agent in chondrocytes and synoviocytes, acting as a new crosstalk factor between these two interconnected events in osteoarthritis. However, a possible function of GRP in the immune system has never been studied. Here we focused our investigation in the involvement of GRP in the cell inflammatory response mechanisms, using a combination of freshly isolated human leucocytes and undifferentiated/differentiated THP-1 cell line. Our results demonstrate that VKDPs such as GRP and matrix gla protein (MGP) are synthesized and γ-carboxylated in the majority of human immune system cells either involved in innate or adaptive immune responses. Stimulation of THP-1 monocytes/macrophages with LPS or hydroxyapatite (HA) up-regulated GRP expression, and treatments with GRP or GRP-coated basic calcium phosphate crystals resulted in the down-regulation of mediators of inflammation and inflammatory cytokines, independently of the protein γ-carboxylation status. Moreover, overexpression of GRP in THP-1 cells rescued the inflammation induced by LPS and HA, by down-regulation of the proinflammatory cytokines TNFα, IL-1β and NFkB. Interestingly, GRP was detected at protein and mRNA levels in extracellular vesicles released by macrophages, which may act as vehicles for extracellular trafficking and release. Our data indicate GRP as an endogenous mediator of inflammatory responses acting as an anti-inflammatory agent in monocytes/macrophages. We propose that in a context of chronic inflammation and calcification-related pathologies, GRP might act as a novel molecular mediator linking inflammation and calcification events, with potential therapeutic application.
Highlights
Inflammation and calcification are common events in chronic inflammatory diseases, such as atherosclerosis and osteoarthritis, involving infiltration of monocytes and accumulation of macrophages [1,2,3,4,5]
Many aspects concerning the molecular mechanisms involved in pathological calcification remain to be elucidated, features such as chronic inflammation, increased extracellular matrix (ECM) remodeling, loss of anticalcific mechanisms leading to proliferation and differentiation of resident cells, and the release of calcifying extracellular vesicles (EVs) are known features contributing to the development of calcific lesions [7,8,9,10,11]
Since Gla-rich protein (GRP) has been previously associated with inflammatory events in chondrocytes and synoviocytes [16], and matrix gla protein (MGP) was recently shown to be up-regulated, together with IL-1β, in RAW 264.7 cells after treatments with vascular smooth muscle cells (VSMCs) conditioned media [6], we studied the GRP is an anti-inflamatory agent in human monocytes/macrophages and MGP production at gene and protein levels. (A) Relative fold expression of GRP and MGP determined by qPCR in isolated leukocytes
Summary
Inflammation and calcification are common events in chronic inflammatory diseases, such as atherosclerosis and osteoarthritis, involving infiltration of monocytes and accumulation of macrophages [1,2,3,4,5]. Many aspects concerning the molecular mechanisms involved in pathological calcification remain to be elucidated, features such as chronic inflammation, increased extracellular matrix (ECM) remodeling, loss of anticalcific mechanisms leading to proliferation and differentiation of resident cells, and the release of calcifying extracellular vesicles (EVs) are known features contributing to the development of calcific lesions [7,8,9,10,11]. Basic calcium phosphate (BCP) crystals, which are the major forms of mineral crystals associated with both atherosclerosis and osteoarthritis, can activate macrophages inducing proinflammatory responses with increased tumor necrosis factor alpha (TNFα), interleukin-(IL) -1β and IL-8 cytokine production [19]. Continuous recycling of the active form of vitamin K is performed by vitamin K epoxide reductase (VKOR), which is inhibited
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