Effects of extracellular osteoanabolic agents on the endogenous response of osteoblastic cells

The complex multidimensional skeletal organization can adapt its structure in accordance with external contexts, demonstrating excellent self-renewal capacity. Thus, optimal extracellular environmental properties are critical for bone regeneration and inextricably linked to the mechanical and biological states of bone. It is interesting to note that the microstructure of bone depends not only on genetic determinants (which control the bone remodeling loop through autocrine and paracrine signals) but also, more importantly, on the continuous response of cells to external mechanical cues. In particular, bone cells sense mechanical signals such as shear, tensile, loading and vibration, and once activated, they react by regulating bone anabolism. Although several specific surrounding conditions needed for osteoblast cells to specifically augment bone formation have been empirically discovered, most of the underlying biomechanical cellular processes underneath remain largely unknown. Nevertheless, exogenous stimuli of endogenous osteogenesis can be applied to promote the mineral apposition rate, bone formation, bone mass and bone strength, as well as expediting fracture repair and bone regeneration. The following review summarizes the latest studies related to the proliferation and differentiation of osteoblastic cells, enhanced by mechanical forces or supplemental signaling factors (such as trace metals, nutraceuticals, vitamins and exosomes), providing a thorough overview of the exogenous osteogenic agents which can be exploited to modulate and influence the mechanically induced anabolism of bone. Furthermore, this review aims to discuss the emerging role of extracellular stimuli in skeletal metabolism as well as their potential roles and provide new perspectives for the treatment of bone disorders

The enzymatic processing of α-dystroglycan by MMP-2 is controlled by two anchoring sites distinct from the active site

Dystroglycan (DG) is a membrane receptor, belonging to the dystrophin-glycoprotein complex (DGC) and formed by two subunits, α-dystroglycan (α-DG) and β-dystroglycan (β -DG). The C-terminal domain of α-DG and the N-terminal extracellular domain of β -DG are connected, providing a link between the extracellular matrix and the cytosol. Under pathological conditions, such as cancer and muscular dystrophies, DG may be the target of metalloproteinases MMP-2 and MMP-9, contributing to disease progression. Previously, we reported that the C-terminal domain α-DG (483-628) domain is particularly susceptible to the catalytic activity of MMP-2; here we show that the α-DG 621-628 region is required to carry out its complete digestion, suggesting that this portion may represent a MMP-2 anchoring site. Following this observation, we synthesized an α-DG based-peptide, spanning the (613-651) C-terminal region. The analysis of the kinetic and thermodynamic parameters of the whole and the isolated catalytic domain of MMP-2 (cdMMP-2) has shown its inhibitory properties, indicating the presence of (at least) two binding sites for the peptide, both located within the catalytic domain, only one of the two being topologically distinct from the catalytic active groove. However, the different behavior between whole MMP-2 and cdMMP-2 envisages the occurrence of an additional binding site for the peptide on the hemopexin-like domain of MMP-2. Interestingly, mass spectrometry analysis has shown that α-DG (613-651) peptide is cleavable even though it is a very poor substrate of MMP-2, a feature that renders this molecule a promising template for developing a selective MMP-2 inhibitor

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

Human matrix metalloproteinases (MMPs) belong to the M10 family of the MA clan of endopeptidases. They are ubiquitarian enzymes, structurally characterized by an active site where a Zn(2+) atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid as a general base. Various MMPs display different domain composition, which is very important for macromolecular substrates recognition. Substrate specificity is very different among MMPs, being often associated to their cellular compartmentalization and/or cellular type where they are expressed. An extensive review of the different MMPs structural and functional features is integrated with their pathological role in several types of diseases, spanning from cancer to cardiovascular diseases and to neurodegeneration. It emerges a very complex and crucial role played by these enzymes in many physiological and pathological processes

Zymographic analysis of EA cells conditioned media exposed to ox-LDL and/or MβCD.

<p>Bands corresponding to the proenzyme forms of gelatinases proMMP-2 (lanes 1–4) and proMMP-9 (lanes 3 and 4) were clearly detected in the conditioned media of treated cells. The gel shown is representing zymograms from 4 independent experiments.</p

Effect of statins on LOX-1 shedding.

<p>(A) Upper panel shows short and long term treatments of LOX-1-V5 COS transfected cells with atorvastatin (2.5 and 5μM) and lovastatin (1.25 and 2.5μM). Densitometric analysis of fold change of protein band of sLOX-1 over respective time matched controls (lower panel). Data represent the averages ± SEM of three experiments. P < 0.05 (*). (B) Statins treatments of HEK-293 #19 and CHO-F2 cells incubated in serum free medium in the presence of atorvastatin (2.5 and 5μM) and lovastatin (1.25μM) for 24h at 37°C.</p

Soluble MMPs mediate LOX-1 shedding.

<p>(A) Fluorescence analysis of LOX-1-V5 transiently transfected COS cells treated without (Ctrl) or 5 mM MβCD or 30μM active MMP-2 or active MMP-1 for 1 h at 37°C. Images show Dil-labelled ox-LDL (red fluorescence) for 1 h at 4°C. Nuclei are blue stained with Hoechst 33342. Scale bar 20 nm. (B) Histogram shows the percentage of red positive cells in different treatments, counting Hoescht-stained nuclei (n≥150). Data represent the average ± SEM of two different experiments.</p

Immunoblotting of EA cells conditioned media probed with anti MMP-1 polyclonal mouse antibodies (upper panel), and TIMP-1 polyclonal mouse antibodies (lower panel).

<p>Optical density analysis was used to semi-quantify the protein secretion levels upon ox-LDL cell-treatment.</p

LOX-1 release in exosomes.

<p>HEK-293#19 cells stably expressing LOX-1 were treated with filipin (5μM) and glimepiride (5μM) for 15, 30 and 60 min. Total protein extract (5μg) was loaded as a positive internal control of electrophoretic mobility (Extr.). Anti-V5 antibody was used to detect LOX-1-V5 protein and antibody directed against flotilin to verify equal protein loading.</p

Effect of receptor-ligand on LOX-1 shedding.

<p>Western blot analysis of media derived from LOX-1-V5 COS transfected cells incubated for 10 min with serum free DMEM (lane 1–3) or conditioned medium derived from EA cells (lane 4–6), in the presence of ox-LDL (A) and atorvastatin (B) at different concentrations, as indicated. Lower panels in (A) and (B) show a 6 times exposure of the gels.</p

sLOX-1 release in COS cells.

<p>(A) LOX-1-V5-COS transfected cells were treated with filipin (5μM) and glimepiride (5μM) for 15, 30 and 60 min. Conditioned media were centrifuged at 100,000 g and the resulting pellets (P100, lanes 2–4) and supernatants (S100, lanes 5–13) were analyzed by Western blotting. Lane 1 (Extr.) shows total protein extract (5 μg) loaded as a positive internal control of electrophoretic mobility of full-length LOX-1 receptor. (B) Upper panel shows sLOX-1 amount released in cells treated with Filipin (5μM) and MβCD (5mM) for 30 min compared to sLOX-1 constitutively released from untreated cells at 1, 2 and 5 hours. Lower panel shows the densitometric analysis for sLOX-1 band intensity. Data in histograms represent the average ± SEM of three experiments, P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***).</p