Abstract
Phytocystatins are endogenous cysteine-protease inhibitors present in plants. They are involved in initial germination rates and in plant defense mechanisms against phytopathogens. Recently, a new phytocystatin derived from sweet orange, CsinCPI-2, has been shown to inhibit the enzymatic activity of human cathepsins, presenting anti-inflammatory potential and pro-osteogenic effect in human dental pulp cells. The osteogenic potential of the CsinCPI-2 protein represents a new insight into plants cysteine proteases inhibitors and this effect needs to be better addressed. The aim of this study was to investigate the performance of pre-osteoblasts in response to CsinCPI-2, mainly focusing on cell adhesion, proliferation and differentiation mechanisms. Together our data show that in the first hours of treatment, protein in CsinCPI-2 promotes an increase in the expression of adhesion markers, which decrease after 24 h, leading to the activation of Kinase-dependent cyclines (CDKs) modulating the transition from G1 to S phases cell cycle. In addition, we saw that the increase in ERK may be associated with activation of the differentiation profile, also observed with an increase in the B-Catenin pathway and an increase in the expression of Runx2 in the group that received the treatment with CsinCPI-2.
Highlights
Bone constantly being in remodeling in a dynamic process requiring coupling of specialized cells and molecules [1], depends on the activity of proteases for breaking-down organic matrix components, such as matrix metaloproteinases (MMP) and cysteine cathepsins, mainly cathepsin K [2,3,4].In turn, the cathepsins degrade the proteins of the extracellular matrix as collagen, laminin, fibronectin and proteoglycans [3] and participate of physiological processes, as tissue remodeling, turnover of the extracellular matrix, inflammation signaling, among others [5,6,7]
To better understand the possible involvement of CsinCPI-2 in the performance of osteoblasts, we investigated different signaling pathways after determining the cytotoxic effect (Fig. 1)
3.1 Modulation of cell proliferation in preosteoblasts responding to CsinCPI-2
Summary
Bone constantly being in remodeling in a dynamic process requiring coupling of specialized cells and molecules [1], depends on the activity of proteases for breaking-down organic matrix components, such as matrix metaloproteinases (MMP) and cysteine cathepsins, mainly cathepsin K [2,3,4].In turn, the cathepsins degrade the proteins of the extracellular matrix as collagen, laminin, fibronectin and proteoglycans [3] and participate of physiological processes, as tissue remodeling, turnover of the extracellular matrix, inflammation signaling, among others [5,6,7]. There are 11 cathepsins encoded in the human genome – B, H, L, S, C, K, O, F, V, X e W [9]; among them, cathepsin K has a key role in bone resorption and is closely related to bone diseases as osteoporosis. The major regulators of the activity of cysteine cathepsins are their own endogenous inhibitors named cystatins [3]. Phytocystatins participate in the regulation of cysteine proteases during programmed cell death, leaf senescence [11] and have been related on control of phytopathogens [11, 12]. The cystatin derived from rice bran can be used in the production of health-promoting bioactive peptides for functional food formulation [16]
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