Prospects and Perspectives for WISP1 (CCN4) in Diabetes Mellitus.

The prevalence of diabetes mellitus (DM) continues to increase throughout the world. In the United States (US) alone, approximately ten percent of the population is diagnosed with DM and another thirty-five percent of the population is considered to have prediabetes. Yet, current treatments for DM are limited and can fail to block the progression of multi-organ failure over time. Wnt1 inducible signaling pathway protein 1 (WISP1), also known as CCN4, is a matricellular protein that offers exceptional promise to address underlying disease progression and develop innovative therapies for DM. WISP1 holds an intricate relationship with other primary pathways of metabolism that include protein kinase B (Akt), mechanistic target of rapamycin (mTOR), AMP activated protein kinase (AMPK), silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1), and mammalian forkhead transcription factors (FoxOs). WISP1 is an exciting prospect to foster vascular as well as neuronal cellular protection and regeneration, control cellular senescence, block oxidative stress injury, and maintain glucose homeostasis. However, under some scenarios WISP1 can promote tumorigenesis, lead to obesity progression with adipocyte hyperplasia, foster fibrotic hepatic disease, and lead to dysregulated inflammation with the progression of DM. Given these considerations, it is imperative to further elucidate the complex relationship WISP1 holds with other vital metabolic pathways to successfully develop WISP1 as a clinically effective target for DM and metabolic disorders.

Recombinant protein CCN5/WISP2 promotes islet cell proliferation and survival in vitro.

Pancreatic ß cell proliferation, survival and function are key elements that need to be considered in developing novel antidiabetic therapies. We recently identified CCN5/WISP2 to have potential growth promoting properties when overexpressed in ß cells; however, further investigations are needed to validate those properties. In this study, we demonstrated that exogenous treatment of insulinoma cells and primary islets with recombinant CCN5 (rh-CCN5) protein enhanced the proliferative capacity which was correlated with activation of cell-cycle regulators CDK4 and cyclin D1. Furthermore, pre-incubation of these cells with rh-CCN5 enhanced their survival rate after being exposed to harsh treatments such as streptozotocin and high concentrations of glucose and free fatty acids. CCN5 as well caused an upregulation in the expression of key genes associated with ß cell identity and function such as GLUT-2 and GCK. Finally, CCN5 activated FAK and downstream ERK kinases which are known to stimulate cell proliferation and survival. Hence, our results validate the growth promoting activities of rh-CCN5 in ß cells and open the door for further investigations in vivo.

CCN5 Reduces Ligamentum Flavum Hypertrophy by Modulating the TGF-ß Pathway.

Ligamentum flavum hypertrophy (LFH) is the most important component of lumbar spinal canal stenosis. Although the pathophysiology of LFH has been extensively studied, no method has been proposed to prevent or treat it. Since the transforming growth factor-ß (TGF-ß) pathway is known to be critical in LFH pathology, we investigated whether LFH could be prevented by blocking or modulating the TGF-ß mechanism. Human LF cells were used for the experiments. First, we created TGF-ß receptor 1 (TGFBR1) knock out (KO) cells with CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 biotechnology and treated them with TGF-ß1 to determine the effects of blocking the TGF-ß pathway. Subsequently, we studied the effect of CCN5, which has recently been proposed to modulate the TGF-ß pathway. To assess the predisposition toward fibrosis, α-smooth muscle actin (αSMA), fibronectin, collagen-1, collagen-3, and CCN2 were evaluated with quantitative real-time polymerase chain reaction, western blotting, and immunocytochemistry. The TGFBR1 KO LF cells were successfully constructed with high KO efficiency. In wild-type (WT) cells, treatment with TGF-ß1 resulted in the overexpression of the messenger RNA (mRNA) of fibrosis-related factors. However, in KO cells, the responses to TGF-ß1 stimulation were significantly lower. In addition, CCN5 and TGF-ß1 co-treatment caused a notable reduction in mRNA expression levels compared with TGF-ß1 stimulation only. The αSMA protein expression increased with TGF-ß1 but decreased with CCN5 treatment. TGF-ß1 induced LF cell transdifferentiation from fibroblasts to myofibroblasts. However, this cell transition dramatically decreased in the presence of CCN5. In conclusion, CCN5 could prevent LFH by modulating the TGF-ß pathway. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2634-2644, 2019.

Cell Biological Assays for Measuring Odontogenic Activities of CCN Proteins.

In postnatal dentin formation, odontoblast differentiation occurs in the pulp tissue regenerative process under pathological condition. Odontoblasts and newly differentiated odontoblast-like cells beneath the caries lesion form tertiary dentin and are highly odontogenic. To observe the activity of dentinogenesis occur within the hard tissue, a combination of immunohistological analysis and immunodetection of dentinogenesis specific molecules, such as dentin sialophosphoprotein (DSPP) and/or its cleaved products dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), is a reliable approach. Besides, recent studies have revealed that the expression of CCN family member 2 (CCN2), a member of the CCN family protein, is confirmed in accordance with tooth development and reparative dentin formation. Therefore, CCN2 could serve as a marker for dentinogenesis. Here, we describe a method for visualizing the CCN2 signal as an odontogenic activity in formalin-fixed paraffin-embedded (FFPE) sections of demineralized human teeth and human dental pulp cells.

Cell Biological Assays for Measuring Angiogenic Activities of CCN Proteins.

Angiogenesis, the process of generating new blood vessels from an existing vasculature, is essential in normal developmental processes such as endochondral ossification and in numerous kinds of pathogenesis including tumor growth. A part from the action of angiogenic factor or antiangiogenic factor, it is still unknown at which stage of the angiogenic cascade these agents affect angiogenesis. Here, we describe methods for the use of connective tissue growth factor (CTGF/CCN2) and CCN2 neutralizing antibody in the currently used principal angiogenesis assays, including those in vitro ones for the proliferation, migration, adhesion, and tube formation of endothelial cells and in vivo assays such as those utilizing type I collagen implantation and the chick chorioallantoic membrane (CAM).

WISP1-αvß3 integrin signaling positively regulates TLR-triggered inflammation response in sepsis induced lung injury.

We recently noted that the matricellular protein WISP1 contributes to sepsis induced acute lung injury (ALI) via integrin ß6. In the current study, we pursued further aspects of WISP1 modulation of TLR signaling in lungs of mice after sepsis and TLR4 mediated release of TNF-α in macrophages. After confirming that TLR4 and CD14 are critical in transducing sepsis mediated ALI, we now demonstrate that intrapulmonary αvß3 is increased by polymicrobrial sepsis in a TLR4, CD14 dependent fashion. Comparison of cultured macrophages revealed that WISP1 increased release of TNF-α from RAW264.7 cells with baseline expression of αvß3, but primary cultures of peritoneal macrophages (PMø) required activation of TLR4 to induce de novo synthesis of αvß3 enabling WISP1 to stimulate release of TNF-α. The specific requirement for ß3 integrin was apparent when the effect of WISP1 was lost in PMø isolated from ß3(-/-) mice. WISP1 enhanced TLR4 mediated ERK signaling and U0126 (an ERK inhibitor) blocked LPS induced ß3 integrin expression and WISP1 enhanced TNF-α release. Collectively these data suggest that WISP1-αvß3 integrin signaling is involved in TLR4 pathways in macrophages and may be an important contributor to TLR4/CD14 mediated inflammation in sepsis induced lung injury.

Wnt2 and WISP-1/CCN4 Induce Intimal Thickening via Promotion of Smooth Muscle Cell Migration.

OBJECTIVE: Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening which acts as a soil for atherosclersosis, as well as causing coronary artery restenosis after stenting and vein graft failure. Investigating factors involved in VSMC migration may enable us to reduce intimal thickening and improve patient outcomes. In this study, we determined whether Wnt proteins regulate VSMC migration and thereby intimal thickening. APPROACH AND RESULTS: Wnt2 mRNA and protein expression were specifically increased in migrating mouse aortic VSMCs. Moreover, VSMC migration was induced by recombinant Wnt2 in vitro. Addition of recombinant Wnt2 protein increased Wnt1-inducible signaling pathway protein-1 (WISP-1) mRNA by ≈1.7-fold, via ß-catenin/T-cell factor signaling, whereas silencing RNA knockdown of Wnt-2 reduced WISP-1 mRNA by ≈65%. Treatment with rWISP-1 significantly increased VSMC migration by ≈1.5-fold, whereas WISP-1 silencing RNA knockdown reduced migration by ≈40%. Wnt2 and WISP-1 effects were integrin-dependent and not additive, indicating that Wnt2 promoted VSMC migration via WISP-1. Additionally, Wnt2 and WISP-1 were significantly increased and colocated in human coronary arteries with intimal thickening. Reduced Wnt2 and WISP-1 levels in mouse carotid arteries from Wnt2(+/-) and WISP-1(-/-) mice, respectively, significantly suppressed intimal thickening in response to carotid artery ligation. In contrast, elevation of plasma WISP-1 via an adenovirus encoding WISP-1 significantly increased intimal thickening by ≈1.5-fold compared with mice receiving control virus. CONCLUSIONS: Upregulation of Wnt2 expression enhanced WISP-1 and promoted VSMC migration and thereby intimal thickening. As novel regulators of VSMC migration and intimal thickening, Wnt2 or WISP-1 may provide a potential therapy for restenosis and vein graft failure.

WISP-1 promotes VEGF-C-dependent lymphangiogenesis by inhibiting miR-300 in human oral squamous cell carcinoma cells.

Oral squamous cell carcinoma (OSCC), which accounts for nearly 90% of head and neck cancers, is characterized by a poor prognosis and a low survival rate. Vascular endothelial growth factor-C (VEGF-C) has been implicated in lymphangiogenesis and is correlated with cancer metastasis. WNT1-inducible signaling pathway protein-1 (WISP)-1/CCN4 is an extracellular matrix-related protein that belongs to the CCN family and stimulates many biological functions. Our previous studies showed that WISP-1 plays an important role in OSCC migration and angiogenesis. However, the effect of WISP-1 on VEGF-C regulation and lymphangiogenesis in OSCC is poorly understood. Here, we showed a correlation between WISP-1 and VEGF-C in tissue specimens from patients with OSCC. To examine the lymphangiogenic effect of WISP-1, we used human lymphatic endothelial cells (LECs) to mimic lymphatic vessel formation. The results showed that conditioned media from WISP-1-treated OSCC cells promoted tube formation and cell migration in LECs. We also found that WISP-1-induced VEGF-C is mediated via the integrin αvß3/integrin-linked kinase (ILK)/Akt signaling pathway. In addition, the expression of microRNA-300 (miR-300) was inhibited by WISP-1 via the integrin αvß3/ILK/Akt cascade. Collectively, these results reveal the detailed mechanism by which WISP-1 promotes lymphangiogenesis via upregulation of VEGF-C expression in OSCC. Therefore, WISP-1 could serve as therapeutic target to prevent metastasis and lymphangiogenesis in OSCC.

WISP-1 contributes to fractionated irradiation-induced radioresistance in esophageal carcinoma cell lines and mice.

Cancer cells that survive fractionated irradiation can be radioresistant and cause tumor recurrence. However, the molecular mechanisms underlying the development of radioresistance in cancer cells remain elusive. The aim of this study was to investigate the role of WISP-1 in the development of radioresistance in esophageal carcinoma during fractionated irradiation. Radioresistant esophageal cancer cells were generated from normal esophageal cancer cells via fractionated irradiation, and expression levels of related proteins were determined by Western blot. Radiosensitivity of cells was established by clonogenic cell survival assays, and cell cycle distribution was evaluated by flow cytometry. Protein distributions were determined by immunofluorescence, and cell toxicity was evaluated by cell counting kit-8 assays. In vivo validations were performed in a xenograft transplantation mouse model. Our data indicate that WISP-1 plays an important role in the development of radioresistance in esophageal cancer cells during fractionated irradiation. The overexression of WISP-1 in esophageal cancer cells was associated with radioresistance. Depletion of extracellular WISP-1 by antibody neutralizing reversed radioresistance and directly induced mitotic catastrophe resulting in cell death. WISP-1 may be a candidate therapeutic target in the treatment of recurrent esophageal carcinoma after radiotherapy.

WISP1 neuroprotection requires FoxO3a post-translational modulation with autoregulatory control of SIRT1.

As a member of the secreted extracellular matrix associated proteins of the CCN family, Wnt1 inducible signaling pathway protein 1 (WISP1/CCN4) is garnering increased attention not only as a potent proliferative entity, but also as a robust cytoprotective agent during toxic insults. Here we demonstrate that WISP1 prevents forkhead transcription factor FoxO3a mediated caspase 1 and caspase 3 apoptotic cell death in primary neurons during oxidant stress. Phosphoinositide 3-kinase (PI 3-K) and protein kinase B (Akt1) are necessary for WISP1 to foster posttranslational phosphorylation of FoxO3a and sequester FoxO3a in the cytoplasm of neurons with protein 14-3-3. Through an autoregulatory loop, WISP1 also minimizes deacytelation of FoxO3a, prevents caspase 1 and 3 activation, and promotes an effective neuroprotective level of SIRT1 activity through SIRT1 nuclear trafficking and prevention of SIRT1 caspase degradation. Elucidation of the critical pathways of WISP1 that determine neuronal cell survival during oxidative stress may offer novel therapeutic avenues for neurodegenerative disorders.