Connective tissue growth factor (CTGF) and cancer progression

Connective tissue growth factor (CTGF) is a member of the CCN family of secreted, matrix-associated proteins encoded by immediate early genes that play various roles in angiogenesis and tumor growth. CCN family proteins share uniform modular structure which mediates various cellular functions such as regulation of cell division, chemotaxis, apoptosis, adhesion, motility, angiogenesis, neoplastic transformation, and ion transport. Recently, CTGF expression has been shown to be associated with tumor development and progression. There is growing body of evidence that CTGF may regulate cancer cell migration, invasion, angiogenesis, and anoikis. In this review, we will highlight the influence of CTGF expression on the biological behavior and progression of various cancer cells, as well as its regulation on various types of protein signals and their mechanisms.     

Estrogen‐related receptor gamma regulates dopaminergic neuronal phenotype by activating GSK3β/NFAT signaling in SH‐SY5Y cells

The orphan nuclear receptor estrogen‐related receptor gamma (ERRγ) is highly expressed in the nervous system during embryogenesis and in adult brains, but its physiological role in neuronal development remains unknown. In this study, we evaluated the relevance of ERRγ in regulating dopaminergic (DAergic) phenotype and the corresponding signaling pathway. We used retinoic acid (RA) to differentiate human neuroblastoma SH‐SY5Y cells. RA induced neurite outgrowth of SH‐SY5Y cells with an increase in DAergic neuron‐like properties, including up‐regulation of tyrosine hydroxylase, dopamine transporter, and vesicular monoamine transporter 2. ERRγ, but not ERRα, was up‐regulated by RA, and participated in RA effect on SH‐SY5Y cells. ERRγ over‐expression enhanced mature DAergic neuronal phenotype with neurite outgrowth as with RA treatment; and RA‐induced increase in DAergic phenotype was attenuated by silencing ERRγ expression. ERRγ appears to have a crucial role in morphological and functional regulation of cells that is selective for DAergic neurons. Polo‐like kinase 2 was up‐regulated in ERRγ‐over‐expressing SH‐SY5Y cells, which was involved in phosphorylation of glycogen synthase kinase 3β and resulting downstream activation of nuclear factor of activated T cells. The likely involvement of ERRγ in regulating the DAergic neuronal phenotype makes this orphan nuclear receptor a novel target for understanding DAergic neuronal differentiation.

     

Role of Smad3, acting independently of transforming growth factor‐β, in the early induction of Wnt‐β‐catenin signaling by parathyroid hormone in mouse osteoblastic cells

Parathyroid hormone (PTH) exerts an anabolic action on bone but the mechanisms are incompletely understood. We showed previously that PTH interacts with the canonical Wnt‐β‐catenin signaling pathway via the transforming growth factor (TGF)‐β signaling molecule, Smad3, to modulate osteoblast differentiation and apoptosis. Here, we examined which actions of Smad3 are TGF‐β‐independent in stimulating the osteoblast phenotype and PTH‐induced Wnt‐β‐catenin signaling. For this, the TGF‐β receptor type 1 [activin receptor‐like kinase (ALK5)] inhibitor (SB431542), and a Smad3 mutant in which the site normally phosphorylated by ALK5 is mutated from SSVS to AAVA, was used. PTH induced total β‐catenin and reduced phosphorylated β‐catenin levels at 1, 6, and 24 h in mouse osteoblastic MC3T3‐E1 cells. Transient transfection of Smad3AAVA inhibited the PTH induction of total β‐catenin and reduction of phosphorylated β‐catenin levels at 6 and 24 h, but not at 1 h, indicating that the early effects occur independently of TGF‐β receptor signaling. On the other hand, MC3T3‐E1 cell clones in which Smad3AAVA was stably expressed demonstrated elevated β‐catenin levels, although alkaline phosphatase (ALP) activity and mineralization were unaltered. In contrast, MC3T3‐E1 cell clones in which wild‐type Smad3 was stably expressed exhibited increased ALP activity and mineralization that were decreased by the ALK5 inhibitor, SB431542, although the β‐catenin levels induced in these cells were not modulated. In conclusion, the present study indicates that PTH induces osteoblast β‐catenin levels via Smad3 independently of, and dependently on, TGF‐β in the early and later induction phases, respectively. J. Cell. Biochem. 108: 285–294, 2009. © 2009 Wiley‐Liss, Inc.     

Connective tissue growth factor (CCN2, CTGF) and organ fibrosis: lessons from transgenic animals

In recent months, four different systems have been reported in the literature in which CCN2 transgenes were individually expressed in podocytes, hepatocytes, cardiomyocytes or respiratory epithelial cells to achieve overexpression in, respectively, the kidney, liver, heart, or lung. These transgenic systems have provided valuable information about the contribution of CCN2 to fibrosis in vivo and have begun to reveal the complexities of the underlying mechanisms involved. On the one hand, studies of these animals have revealed that CCN2 overexpression does not necessarily lead directly to fibrotic pathology but may cause severe non-fibrotic tissue damage due to its other effects on cell function (e.g. heart). On the other hand, overexpression of CCN2 in concert with signaling pathways associated with development (e.g. lung) or fibrosing injuries (e.g. kidney, liver) can lead to the initiation or exacerbation of fibrosis. The significance of these studies is discussed in the context of the requirement for interactions between CCN2 and co-stimulatory factors in the microenvironment for the manifestation of CCN2-dependent fibrosis.     

Decrease of GSK3β phosphorylation in the rat nucleus accumbens core enhances cocaine‐induced hyper‐locomotor activity

Glycogen synthase kinase 3β (GSK3β), which is abundantly present in the brain, is known to contribute to psychomotor stimulant‐induced locomotor behaviors. However, most studies have been focused in showing that GSK3β is able to attenuate psychomotor stimulants‐induced hyperactivity by increasing its phosphorylation levels in the nucleus accumbens (NAcc). So, here we examined in the opposite direction about the effects of decreased phosphorylation of GSK3β in the NAcc core on both basal and cocaine‐induced locomotor activity by a bilateral microinjection into this site of an artificially synthesized peptide, S9 (0.5 or 5.0 μg/μL), which contains sequences around N‐terminal serine 9 residue of GSK3β. We found that decreased levels of GSK3β phosphorylation in the NAcc core enhance cocaine‐induced hyper‐locomotor activity, while leaving basal locomotor activity unchanged. This is the first demonstration, to our knowledge, that the selective decrease of GSK3β phosphorylation levels in the NAcc core may contribute positively to cocaine‐induced locomotor activity, while this is not sufficient for the generation of locomotor behavior by itself without cocaine. Taken together, these findings importantly suggest that GSK3β may need other molecular targets which are co‐activated (or deactivated) by psychomotor stimulants like cocaine to contribute to generation of locomotor behaviors.     

PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells

Protein arginine methyltransferase 5 (PRMT5) has been implicated in the development and progression of human cancers. However, few studies reveal its role in epithelial‐mesenchymal transition (EMT) of pancreatic cancer cells. In this study, we find that PRMT5 is up‐regulated in pancreatic cancer, and promotes proliferation, migration and invasion in pancreatic cancer cells, and promotes tumorigenesis. Silencing PRMT5 induces epithelial marker E‐cadherin expression and down‐regulates expression of mesenchymal markers including Vimentin, collagen I and β‐catenin in PaTu8988 and SW1990 cells, whereas ectopic PRMT5 re‐expression partially reverses these changes, indicating that PRMT5 promotes EMT in pancreatic cancer. More importantly, we find that PRMT5 knockdown decreases the phosphorylation level of EGFR at Y1068 and Y1172 and its downstream p‐AKT and p‐GSK3β, and then results in down‐regulation of β‐catenin. Expectedly, ectopic PRMT5 re‐expression also reverses the above changes. It is suggested that PRMT5 promotes EMT probably via EGFR/AKT/β‐catenin pathway. Taken together, our study demonstrates that PRMT5 plays oncogenic roles in the growth of pancreatic cancer cell and provides a potential candidate for pancreatic cancer treatment.     

Inhibition of GSK‐3β enhances neural differentiation in unrestricted somatic stem cells

GSK‐3β is a key molecule in several signalling pathways, including the Wnt/β‐catenin signalling pathway. There is increasing evidence suggesting Wnt/β‐catenin signalling is involved in the neural differentiation of embryonic, somatic and neural stem cells. However, a large body of evidence indicates that this pathway maintains stem cells in a proliferative state. To address this controversy, we have investigated whether the Wnt/β‐catenin pathway is present and involved in the neural differentiation of newly introduced USSCs (unrestricted somatic stem cells). Our results indicate that the components of Wnt/β‐catenin signalling are present in undifferentiated USSCs. We also show that the treatment of neurally induced USSCs with BIO (6‐bromoindirubin‐3′‐oxime), a specific GSK‐3β inhibitor and Wnt activator, for 5 and 10 days results in increased expression of a general neuronal marker (β‐tubulin III). Moreover, the expression of pGSK‐3β and stabilized β‐catenin increased by BIO in neurally induced USSCs, indicates that the Wnt pathway is activated and functional in these cells. Thus, inhibition of GSK‐3β in USSCs enhances their neural differentiation, which suggests a positive role of the Wnt/β‐catenin signalling pathway towards neural fate.     

Wnt signaling loss accelerates the appearance of neuropathological hallmarks of Alzheimer's disease in J20‐APP transgenic and wild‐type mice

Alzheimer's disease (AD ) is a neurodegenerative pathology characterized by aggregates of amyloid‐β (Aβ) and phosphorylated tau protein, synaptic dysfunction, and spatial memory impairment. The Wnt signaling pathway has several key functions in the adult brain and has been associated with AD , mainly as a neuroprotective factor against Aβ toxicity and tau phosphorylation. However, dysfunction of Wnt/β‐catenin signaling might also play a role in the onset and development of the disease. J20 APP swInd transgenic (Tg) mouse model of AD was treated i.p. with various Wnt signaling inhibitors for 10 weeks during pre‐symptomatic stages. Then, cognitive, biochemical and histochemical analyses were performed. Wnt signaling inhibitors induced severe changes in the hippocampus, including alterations in Wnt pathway components and loss of Wnt signaling function, severe cognitive deficits, increased tau phosphorylation and Aβ_1–42 peptide levels, decreased Aβ42/Aβ40 ratio and Aβ_1–42 concentration in the cerebral spinal fluid, and high levels of soluble Aβ species and synaptotoxic oligomers in the hippocampus, together with changes in the amount and size of senile plaques. More important, we also observed severe alterations in treated wild‐type (WT ) mice, including behavioral impairment, tau phosphorylation, increased Aβ_1–42 in the hippocampus, decreased Aβ_1–42 in the cerebral spinal fluid, and hippocampal dysfunction. Wnt inhibition accelerated the development of the pathology in a Tg AD mouse model and contributed to the development of Alzheimer's‐like changes in WT mice. These results indicate that Wnt signaling plays important roles in the structure and function of the adult hippocampus and suggest that inhibition of the Wnt signaling pathway is an important factor in the pathogenesis of AD .

Read the Editorial Highlight for this article on page 356 .

     

β‐arrestin2/miR‐155/GSK3β regulates transition of 5′‐azacytizine‐induced Sca‐1‐positive cells to cardiomyocytes

Stem‐cell antigen 1–positive (Sca‐1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5′‐azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. β‐arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of β‐arrestin2 in Sca‐1+ CSC differentiation, we used β‐arrestin2–knockout mice and overexpression strategies. Real‐time PCR revealed that β‐arrestin2 promoted 5′‐azacytizine‐induced Sca‐1+ CSC differentiation in vitro. Because the microRNA 155 (miR‐155) may regulate β‐arrestin2 expression, we detected its role and relationship with β‐arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR‐155. Real‐time PCR revealed that miR‐155, inhibited by β‐arrestin2, impaired 5′‐azacytizine‐induced Sca‐1+ CSC differentiation. On luciferase report assay, miR‐155 could inhibit the activity of β‐arrestin2 and GSK3β, which suggests a loop pathway between miR‐155 and β‐arrestin2. Furthermore, β‐arrestin2‐knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in β‐arrestin2‐Knockout mice, so the activity of GSK3β was regulated by β‐arrestin2 not Akt. We transplanted Sca‐1+ CSCs from β‐arrestin2‐knockout mice to mice with myocardial infarction and found similar protective functions as in wild‐type mice but impaired arterial elastance. Furthermore, low level of β‐arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β‐arrestin2/miR‐155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.     

Lithium induces follicular atresia in rat ovary through a GSK‐3β/β‐catenin dependent mechanism

Lithium chloride (LiCl) is a drug used to treat bipolar disorder, but has side effects in the female reproductive system. Although lithium is known to decrease folliculogenesis and induce follicular atresia in rodent ovaries, its cellular and molecular effects in the ovary have not yet been addressed. To investigate these effects, 23‐day‐old immature female rats were injected with 10 IU pregnant mare serum gonadotropin (PMSG), followed by injections of 250 mg/kg LiCl every 12 hr for four doses. Ovaries were removed 40 and 48 hr after PMSG administration and prepared for histology, immunohistochemistry, Western blotting, and DNA laddering analysis. Our results showed that in the ovaries of LiCl‐treated rats, few antral but more atretic follicles were present compared to those of the control rats. The induction of atresia by LiCl was further confirmed by the presence of DNA fragmentation, accompanied by a reduced level of 17β‐estradiol in the serum. At the cellular level, lithium significantly decreased the number of proliferating cell nuclear antigen (PCNA)‐positive cells and conversely increased the number of TUNEL‐positive cells in the granulosa layer of the antral follicles. At the molecular level, lithium increased the level of phosphorylated glycogen synthase kinase‐3β, and unexpectedly decreased the expression of active (stabilized) β‐catenin. Altogether, our results indicate that lithium disrupts the balance between proliferation and apoptosis in granulosa cells, leading to follicular atresia possibly through the reduction in both the stabilized β‐catenin and 17β‐estradiol synthesis. Mol. Reprod. Dev. 80: 286–296, 2013. © 2013 Wiley Periodicals, Inc.     

Identification of glycogen synthase kinase 3α as a therapeutic target in melanoma

Deregulated expression or activity of kinases can lead to melanomas, but often the particular kinase isoform causing the effect is not well established, making identification and validation of different isoforms regulating disease development especially important. To accomplish this objective, an siRNA screen was undertaken that which identified glycogen synthase kinase 3α (GSK3α) as an important melanoma growth regulator. Melanocytes and melanoma cell lines representing various stages of melanoma tumor progression expressed both GSK3α and GSK3β, but analysis of tumors in patients with melanoma showed elevated expression of GSK3α in 72% of samples, which was not observed for GSK3β. Furthermore, 80% of tumors in patients with melanoma expressed elevated levels of catalytically active phosphorylated GSK3α (pGSK3αY279), but not phosphorylated GSK3β (pGSK3βY216). siRNA‐mediated reduction in GSK3α protein levels reduced melanoma cell survival and proliferation, sensitized cells to apoptosis‐inducing agents and decreased xenografted tumor development by up to 56%. Mechanistically, inhibiting GSK3α expression using siRNA or the pharmacological agent AR‐A014418 arrested melanoma cells in the G0/G1 phase of the cell cycle and induced apoptotic death to retard tumorigenesis. Therefore, GSK3α is a key therapeutic target in melanoma.     

Phosphorylation of PPP(S/T)P motif of the free LRP6 intracellular domain is not required to activate the Wnt/β‐catenin pathway and attenuate GSK3β activity

The canonical Wnt/β‐catenin signaling pathway plays a critical role in numerous physiological and pathological processes. LRP6 is an essential co‐receptor for Wnt/β‐catenin signaling; as transduction of the Wnt signal is strongly dependent upon GSK3β‐mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane‐anchored LRP6 intracellular domain. Previously, we showed that the free LRP6 intracellular domain (LRP6‐ICD) can activate the Wnt/β‐catenin pathway in a β‐catenin and TCF/LEF‐1 dependent manner, as well as interact with and attenuate GSK3β activity. However, it is unknown if the ability of LRP6‐ICD to attenuate GSK3β activity and modulate activation of the Wnt/β‐catenin pathway requires phosphorylation of the LRP6‐ICD PPP(S/T)P motifs, in a manner similar to the membrane‐anchored LRP6 intracellular domain. Here we provide evidence that the LRP6‐ICD does not have to be phosphorylated at its PPP(S/T)P motif by GSK3β to stabilize endogenous cytosolic β‐catenin resulting in activation of TCF/LEF‐1 and the Wnt/β‐catenin pathway. LRP6‐ICD and a mutant in which all 5 PPP(S/T)P motifs were changed to PPP(A)P motifs equivalently interacted with and attenuated GSK3β activity in vitro, and both constructs inhibited the in situ GSK3β‐mediated phosphorylation of β‐catenin and tau to the same extent. These data indicate that the LRP6‐ICD attenuates GSK3β activity similar to other GSK3β binding proteins, and is not a result of it being a GSK3β substrate. Our findings suggest the functional and regulatory mechanisms governing the free LRP6‐ICD may be distinct from membrane‐anchored LRP6, and that release of the LRP6‐ICD may provide a complimentary signaling cascade capable of modulating Wnt‐dependent gene expression. J. Cell. Biochem. 108: 886–895, 2009. © 2009 Wiley‐Liss, Inc.