Fayez F. Safadi

Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/ CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation

Pre-osteoblast adhesion and interaction with extracellular matrix (ECM) proteins through integrin receptors result in activation of signaling pathways regulating osteoblast differentiation. Connective tissue growth factor (CTGF/CCN2) is a matricellular protein secreted into the ECM. Prior studies in various cell types have shown that cell adhesion to CTGF via integrin receptors results in activation of specific signaling pathways that regulate cell functions, such as differentiation and cytoskeletal reorganization. To date, there are no studies that have examined whether CTGF can serve as an adhesive substrate for osteoblasts. In this study, we used the MC3T3-E1 cell line to demonstrate that CTGF serves as an adhesive matrix for osteoblasts. Anti-integrin blocking experiments and co-immunoprecipitation assays demonstrated that the integrin αvβ1 plays a key role in osteoblast adhesion to a CTGF matrix. Immunofluorescence staining of osteoblasts cultured on a CTGF matrix confirmed actin cytoskeletal reorganization, enhanced spreading, formation of focal adhesions, and activation of Rac1. Alkaline phosphatase (ALP) staining and activity assays, as well as Alizarin red staining demonstrated that osteoblast attachment to CTGF matrix enhanced maturation, bone nodule formation and matrix mineralization. To investigate whether the effect of CTGF on osteoblast differentiation involves integrin-mediated activation of specific signaling pathways, we performed Western blot, chromatin immunoprecipitation (ChIP) and qPCR assays. Osteoblasts cultured on a CTGF matrix showed increased total and phosphorylated (activated) forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Inhibition of ERK blocked osteogenic differentiation in cells cultured on a CTGF matrix. There was an increase in runt-related transcription factor 2 (Runx2) binding to the osteocalcin gene promoter, and in the expression of osteogenic markers regulated by Runx2. Collectively, the results of this study are the first to demonstrate CTGF serves as a suitable matrix protein, enhancing osteoblast adhesion (via αvβ1 integrin) and promoting cell spreading via cytoskeletal reorganization and Rac1 activation. Furthermore, integrin-mediated activation of ERK signaling resulted in increased osteoblast differentiation accompanied by an increase in Runx2 binding to the osteocalcin promoter and in the expression of osteogenic markers.

The Effect of Class A Scavenger Receptor Deficiency in Bone

Class A scavenger receptor (SR-A) is predominantly expressed by macrophages, and because osteoclasts are of monocyte/macrophage lineage, SR-A is of potential interest in osteoclast biology. In addition to modified low density lipoprotein uptake, SR-A is also important in cell attachment and signaling. In this study we evaluated the effect of SR-A deletion on bone. Knock-out animals have 40% greater body weight than wild type. Body composition analyses demonstrated that total lean and fat body mass were greater in knock-out animals, but there was no significant difference in percent fat and lean body mass. Bone mineral density and content were significantly greater in knock-out compared with wild type animals. Micro-computed tomography analyses confirmed that total volume, bone volume as well as trabecular number, thickness, and connectivity were significantly greater in knock-out mice. As expected, trabecular separation was greater in wild type mice. The phenotype appears to be explained by 60% fewer osteoclasts in females and 35% fewer in males compared to wild type mice with a paradoxical increase in nuclei/osteoclast in knock-out animals. Furthermore, there were no differences in adipocyte number and osteoblast number or activity. The addition of the soluble extracellular domain of SR-A to RAW264.7 cells stimulated a concentration-dependent increase in osteoclast differentiation that was receptor activator of nuclear factor-κB ligand (RANKL)-dependent. Soluble SR-A had no effect on cell proliferation in the presence of RANKL but stimulated a 40% increase in numbers in the absence of RANKL. We conclude that SR-A plays a role in normal osteoclast differentiation, suggesting a novel role for this receptor in bone biology.

Mutation in Osteoactivin Promotes Receptor Activator of NFκB Ligand (RANKL)-mediated Osteoclast Differentiation and Survival but Inhibits Osteoclast Function.

Background: The importance of osteoactivin has emerged from its role in osteogenesis. Results: Loss-of-function mutation of osteoactivin stimulates osteoclast differentiation and survival, with a defect in bone resorption. Conclusion: Osteoactivin acts as a negative regulator of osteoclastogenesis and a positive regulator of osteoclast function. Significance: This study helps to identify the possible mechanisms and targets of osteoactivin during osteoclast differentiation with impact on bone remodeling. We previously reported on the importance of osteoactivin (OA/Gpnmb) in osteogenesis. In this study, we examined the role of OA in osteoclastogenesis, using mice with a nonsense mutation in the Gpnmb gene (D2J) and wild-type controls (D2J/Gpnmb+). In these D2J mice, micro-computed tomography and histomorphometric analyses revealed increased cortical thickness, whereas total porosity and eroded surface were significantly reduced in D2J mice compared with wild-type controls, and these results were corroborated by lower serum levels of CTX-1. Contrary to these observations and counterintuitively, temporal gene expression analyses supported up-regulated osteoclastogenesis in D2J mice and increased osteoclast differentiation rates ex vivo, marked by increased number and size. The finding that MAPK was activated in early differentiating and mature D2J osteoclasts and that survival of D2J osteoclasts was enhanced and mediated by activation of the AKT-GSK3β pathway supports this observation. Furthermore, this was abrogated by the addition of recombinant OA to cultures, which restored osteoclastogenesis to wild-type levels. Moreover, mix and match co-cultures demonstrated an induction of osteoclastogenesis in D2J osteoblasts co-cultured with osteoclasts of D2J or wild-type. Last, in functional osteo-assays, we show that bone resorption activity of D2J osteoclasts is dramatically reduced, and these osteoclasts present an abnormal ruffled border over the bone surface. Collectively, these data support a model whereby OA/Gpnmb acts as a negative regulator of osteoclast differentiation and survival but not function by inhibiting the ERK/AKT signaling pathways.

Osteoactivin Promotes Migration of Oral Squamous Cell Carcinomas

Nearly 50% of patients with oral squamous cell carcinoma (OSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell adhesion, migration, and invasion. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies. The aims were to determine how integrin interactions modulate OA‐induced OSCC cell migration; and to investigate OA effects on cell survival and proliferation. We confirmed OA mRNA and protein overexpression in OSCC cell lines. We assessed OAs interactions with integrins using adhesion inhibition assays, fluorescent immunocytochemistry and co‐immunoprecipitation. We investigated OA‐mediated activation of mitogen‐activated protein kinases (MAPKs) and cell survival. Integrin inhibition effects on OA‐mediated cell migration were determined. We assessed effects of OA knock‐down on cell migration and proliferation. OA is overexpressed in OSCC cell lines, and serves as a migration‐promoting adhesion molecule. OA co‐localized with integrin subunits, and co‐immunoprecipitated with the subunits. Integrin blocking antibodies, especially those directed against the β1 subunit, inhibited cell adhesion (P = 0.03 for SCC15 cells). Adhesion to OA activated MAPKs in UMSCC14a cells and OA treatment promoted survival of SCC15 cells. Integrin‐neutralizing antibodies enhanced cell migration with OA in the extracellular matrix. OA knock‐down resulted in decreased proliferation of SCC15 and SCC25 cells, but did not inhibit cell migration. OA in the extracellular matrix promotes OSCC cell adhesion and migration, and may be a novel target in the prevention of HNSCC spread. J. Cell. Physiol. 231: 1761–1770, 2016.

Parkinson's disease biomarker: a patent evaluation of WO2013153386.

Introduction: Parkinson’s disease (PD) is a neurodegenerative movement disorder resultant from the loss of dopaminergic neurons in the brain. There is an urgent need for effective biomarkers that can be used in the early diagnosis of PD. Mitochondrial dysfunction plays a significant role in PD pathology, which has led to the evaluation of mitophagy markers, PTEN-induced putative kinase 1 (PINK1), and PARKIN as possible biomarkers for the early diagnosis of PD. Areas covered: The current patent describes the use of phosphorylation of PINK1 and PARKIN as a diagnostic measure. Specifically, Ser65 on PARKIN, which is phosphorylated by PINK1, and the autophosphorylation of PINK1 at Thr257 are described. Expert opinion: This patent describes a much needed methodology that can easily be adapted in the clinical setting by which a biological sample, such as serum or cerebrospinal fluid, is collected and analyzed for the phosphorylation markers. Here, the phosphorylation activity seen in PINK1 and PARKIN can differentiate between age-matched controls and PD patients. This patent presents a novel diagnostic measure in early PD, as well as determines which medications would have a beneficial effect on a patient’s disease progression.

The glycoprotein GPNMB attenuates astrocyte inflammatory responses through the CD44 receptor

BackgroundNeuroinflammation is one of the hallmarks of neurodegenerative diseases, such as Parkinson’s disease (PD). Activation of glial cells, including microglia and astrocytes, is a characteristic of the inflammatory response. Glycoprotein non-metastatic melanoma protein B (GPNMB) is a transmembrane glycoprotein that releases a soluble signaling peptide when cleaved by ADAM10 or other extracellular proteases. GPNMB has demonstrated a neuroprotective role in animal models of ALS and ischemia. However, the mechanism of this protection has not been well established. CD44 is a receptor expressed on astrocytes that can bind GPNMB, and CD44 activation has been demonstrated to reduce NFκB activation and subsequent inflammatory responses in macrophages. GPNMB signaling has not been investigated in models of PD or specifically in astrocytes. More recently, genetic studies have linked polymorphisms in GPNMB with risk for PD. Therefore, it is important to understand the role this signaling protein plays in PD.MethodsWe used data mining techniques to evaluate mRNA expression of GPNMB and its receptor CD44 in the substantia nigra of PD and control brains. Immunofluorescence and qPCR techniques were used to assess GPNMB and CD44 levels in mice treated with MPTP. In vitro experiments utilized the immortalized mouse astrocyte cell line IMA2.1 and purified primary mouse astrocytes. The effects of recombinant GPNMB on cytokine-induced astrocyte activation was determined by qPCR, immunofluorescence, and measurement of nitric oxide and reactive oxygen production.ResultsIncreased GPNMB and CD44 expression was observed in the substantia nigra of human PD brains and in GFAP-positive astrocytes in an animal model of PD. GPNMB treatment attenuated cytokine-induced levels of inducible nitric oxide synthase, nitric oxide, reactive oxygen species, and the inflammatory cytokine IL-6 in an astrocyte cell line and primary mouse astrocytes. Using primary mouse astrocytes from CD44 knockout mice, we found that the anti-inflammatory effects of GPNMB are CD44-mediated.ConclusionsThese results demonstrate that GPNMB may exert its neuroprotective effect through reducing astrocyte-mediated neuroinflammation in a CD44-dependent manner, providing novel mechanistic insight into the neuroprotective properties of GPNMB.

Osteoactivin regulates head and neck squamous cell carcinoma invasion by modulating matrix metalloproteases

Nearly 60% of patients with head and neck squamous cell carcinoma (HNSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell migration and invasion, which are in part dependent on extracellular matrix degradation by matrix metalloproteinases. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies, and has been shown to upregulate matrix metalloproteinase (MMP) expression and activity. To determine how OA modulates MMP expression and activity in HNSCC, and to investigate OA effects on cell invasion, we assessed effects of OA treatment on MMP mRNA and protein expression, as well as gelatinase and caseinolytic activity in HNSCC cell lines. We assessed the effects of OA gene silencing on MMP expression, gelatinase and caseinolytic activity, and cell invasion. OA treatment had differential effects on MMP mRNA expression. OA treatment upregulated MMP‐10 expression in UMSCC14a (p = 0.0431) and SCC15 (p < 0.0001) cells, but decreased MMP‐9 expression in UMSCC14a cells (p = 0.0002). OA gene silencing decreased MMP‐10 expression in UMSCC12 cells (p = 0.0001), and MMP‐3 (p = 0.0005) and ‐9 (p = 0.0036) expression in SCC25 cells. In SCC15 and SCC25 cells, OA treatment increased MMP‐2 (p = 0.0408) and MMP‐9 gelatinase activity (p < 0.0001), respectively. OA depletion decreased MMP‐2 (p = 0.0023) and ‐9 (p < 0.0001) activity in SCC25 cells. OA treatment increased 70 kDa caseinolytic activity in UMSCC12 cells consistent with tissue type plasminogen activator (p = 0.0078). OA depletion decreased invasive capacity of UMSCC12 cells (p < 0.0001). OAs effects on MMP expression in HNSCC are variable, and may promote cancer cell invasion.

TRAPPC9: Novel insights into its trafficking and signaling pathways in health and disease (Review)

Trafficking protein particle complex 9 (TRAPPC9) is a protein subunit of the transport protein particle II (TRAPPII), which has been reported to be important in the trafficking of cargo from the endoplasmic reticulum (ER) to the Golgi, and in intra‑Golgi and endosome‑to‑Golgi transport in yeast cells. In mammalian cells, TRAPPII has been shown to be important in Golgi vesicle tethering and intra‑Golgi transport. TRAPPC9 is considered to be a novel molecule capable of modulating the activation of nuclear factor‑κB (NF‑κB). Mutations in TRAPPC9 have been linked to a rare consanguineous hereditary form of mental retardation, as part of the NF‑κB pathways. In addition, TRAPPC9 has been reported to be involved in breast and colon cancer and liver diseases. The present review highlights the most recent publications on the structure, expression and function of TRAPPC9, and its association with various human diseases.