Hyaluronan injection in murine osteoarthritis prevents TGFbeta 1-induced synovial neovascularization and fibrosis and maintains articular cartilage integrity by a CD44-dependent mechanism

Introduction

The goals of this study were to examine the oxemic regulation of Wnt signaling to explore whether Wnt signaling accelerates the age-related degeneration of nucleus pulposus cells, and if so, to define the mechanism underlying this effect. We investigated the expression of Klotho, a newly identified antiaging gene, and whether its regulation is attributable to the suppression of Wnt signaling.

Methods

Rat nucleus pulposus cells were cultured under normoxic (21% O₂) or hypoxic (2% O₂) conditions, and the expression and promoter activity of Wnt signaling and Klotho were evaluated. The effect of Klotho protein was examined with transfection experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, senescence-associated ??-galactosidase staining, and cell-cycle analysis. To determine the methylation status of the Klotho promoter region, bisulfite genomic sequencing analysis was performed. Its relation with the activation of Wnt signaling was assessed. We also examined whether the expression of Klotho could block the effects of pathological Wnt expression in nucleus pulposus cells.

Results

Nucleus pulposus cells exhibited increased ??-catenin mRNA and protein under the hypoxic condition. Klotho protein was expressed in vivo, and protein and messenger RNA expression decreased under the hypoxic condition. Klotho treatment decreased cell proliferation and induced the quiescence of nucleus pulposus cells. In addition, Klotho treatment inhibited expression of ??-catenin gene and protein compared with untreated control cells.

Conclusions

These data indicate that Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells. These results suggest that the expression of Klotho is regulated by the balance between upregulation and downregulation of Wnt signaling.     

Activation of nuclear factor kappa B (NF-κB) by connective tissue growth factor (CCN2) is involved in sustaining the survival of primary rat hepatic stellate cells

Background

Secreted Frizzled related proteins (SFRPs) are extracellular regulators of Wnt signaling. These proteins contain an N-terminal cysteine rich domain (CRD) highly similar to the CRDs of the Frizzled family of seven-transmembrane proteins that act as Wnt receptors. SFRPs can bind to Wnts and prevent their interaction with the Frizzled receptor. Recently it has been reported that a splice variant of human Frizzled-4 (FZD4S) lacking the transmembrane and the cytoplasmic domains of Frizzled-4 can activate rather than inhibit Wnt-8 activity in Xenopus embryos. This indicates that secreted CRD containing proteins such as Frizzled ecto-domains and SFRPs may not always act as Wnt inhibitors. It is not known how FZD4S can activate Wnt/β-catenin signaling and what biological role this molecule plays in vivo.

Results

Here we report that the Xenopus frizzled-4 is alternatively spliced to give rise to a putative secreted protein that lacks the seven-transmembrane and the cytoplasmic domains. We performed functional experiments in Xenopus embryos to investigate how this novel splicing variant, Xfz4S, can modulate the Wnt/β-catenin pathway. We show that Xfz4S as well as the extracellular domain of Xfz8 (ECD8) can act as both activators and inhibitors of Wnt/β-catenin signaling dependent on the Wnt ligand presented. The positive regulation of Wnt/β-catenin signaling by the extracellular domains of Frizzled receptors is mediated by the members of low density lipoprotein receptor-related protein (LRP-5/6) that act as Wnt coreceptors.

Conclusion

This work provides evidence that the secreted extracellular domains of Frizzled receptors may act as both inhibitors and activators of Wnt signaling dependent on the Wnt ligand presented.     

Discovery and Characterization of a Small Molecule Inhibitor of the PDZ Domain of Dishevelled

Dishevelled (Dvl) is an essential protein in the Wnt signaling pathways; it uses its PDZ domain to transduce the Wnt signals from the membrane receptor Frizzled to downstream components. Here, we report identifying a drug-like small molecule compound through structure-based ligand screening and NMR spectroscopy and show the compound to interact at low micromolar affinity with the PDZ domain of Dvl. In a Xenopus testing system, the compound could permeate the cell membrane and block the Wnt signaling pathways. In addition, the compound inhibited Wnt signaling and reduced the levels of apoptosis in the hyaloid vessels of eye. Moreover, this compound also suppressed the growth of prostate cancer PC-3 cells. These biological effects suggest that by blocking the PDZ domain of Dvl, the compound identified in our studies effectively inhibits the Wnt signaling and thus provides a useful tool for studies dissecting the Wnt signaling pathways.The Wnt signaling pathways are regulated by a family of secreted Wnt glycoproteins. The canonical Wnt pathway, which is highly conserved, is best understood. In this pathway, Wnt molecules interact with the seven-transmembrane Frizzled (Fz)² proteins (1) by binding to an N-terminal cysteine-rich-domain (2). The signal is then transduced into the cell through an internal sequence of Fz, C-terminal to the seventh transmembrane domain, which binds directly to the PDZ (postsynaptic density-95/discs large/zonula occludens-1) domain of the cytoplasmic protein Dishevelled (Dvl) (3). Dvl then transduces the Wnt signals to downstream components (4). Three Dvl homologs (Dvl-1, -2, and -3) have been identified in humans; all are expressed in both embryonic and adult tissues, including brain, heart, lung, kidney, skeletal muscle, and others (4). Up-regulation and overexpression of Dvl proteins have been reported in many cancers, including those of breast, colon, prostate, mesothelium, and lung (non-small cell) (5–8).The Dvl protein is made up of three conserved domains: an N-terminal DIX domain, a central PDZ domain, and a C-terminal DEP domain (9). The central PDZ domain is of particular interest because of its interaction with Fz and other Wnt pathway proteins (3, 10). The direct interaction between the PDZ domain and Fz peptides is relatively weak, and other factors may play a role to ensure the communication between the two molecules (3). For example, several studies suggest that the DEP domain of Dvl has a membrane-targeting function that may facilitate PDZ-Fz interaction (11–14). However, the weak PDZ-Fz interaction provides an opportunity to block Wnt signaling at the Dvl level by using a small molecule inhibitor. An earlier study in our laboratories used an NMR-assisted virtual ligand screening approach to identify a peptide mimic that can bind to the Dvl PDZ domain (15). We have now used an improved algorithm to conduct an additional structure-based virtual screen of the PDZ domain of Dvl and have discovered a group of drug-like compounds that bind to the PDZ domain with moderate to low micromolar affinity. One of these compounds effectively blocked Wnt signaling in vivo and reduced the growth rate of a prostate cancer cell line.     

Ror2/Frizzled Complex Mediates Wnt5a-Induced AP-1 Activation by Regulating Dishevelled Polymerization

The receptor tyrosine kinase Ror2 acts as a receptor or coreceptor for Wnt5a to mediate Wnt5a-induced activation of the Wnt/JNK pathway and inhibition of the β-catenin-dependent canonical Wnt pathway. However, little is known about how Ror2 cooperates with another receptor component(s) to mediate Wnt5a signaling. We show here that Ror2 regulates Wnt5a-induced polymerization of Dishevelled (Dvl) and that this Ror2-mediated regulation of Dvl is independent of the cytoplasmic region of Ror2. Ror2 can associate with Frizzled7 (Fz7) via its extracellular cysteine-rich domain to form a receptor complex that is required for the regulation of Dvl and activation of the AP-1 promoter after Wnt5a stimulation. Suppressed expression of Fz7 indeed results in the inhibition of Wnt5a-induced polymerization of Dvl and AP-1 activation. Interestingly, both the DIX and the DEP domains of Dvl are indispensable for Dvl polymerization and subsequent AP-1 activation after Wnt5a stimulation. We further show that polymerized Dvl is colocalized with Rac1 and that suppressed expression of Rac1 inhibits Wnt5a-induced AP-1 activation. Collectively, our results indicate that Ror2/Fz receptor complex plays an important role in the Wnt5a/Rac1/AP-1 pathway by regulating the polymerization of Dvl.Wnt proteins can elicit β-catenin-dependent and -independent signaling pathways (2, 20, 46). Ror2 is a member of the Ror family of receptor tyrosine kinases and plays essential roles in developmental morphogenesis (21, 26, 31, 32, 44). Ror2 has been shown to act as a receptor or coreceptor for Wnt5a to activate the β-catenin-independent signaling pathway, involving JNK/c-Jun (AP-1), Src and Ca²⁺, which are essential for cell polarity, migration, and cancer cell invasion (8, 14, 28-31, 37). Wnt5a/Ror2 signaling also plays a crucial role in inhibiting the β-catenin-dependent signaling pathway (25). Structure-function analyses of Ror2 revealed that Ror2 mediates Wnt5a signaling through distinct mechanisms dependent on and independent of its kinase activity, i.e., Wnt5a-induced migration of fibroblast cells requires the cytoplasmic C-terminal portion of Ror2 but not its intrinsic kinase activity (28), whereas the intrinsic kinase activity of Ror2 is indispensable for extracellular matrix (ECM) degradation of osteosarcoma cells (8). In addition, inhibition of the β-catenin-dependent signaling pathway by Wnt5a also requires the intrinsic kinase activity of Ror2 (24). Importantly, the Caenorhabditis elegans ortholog of Ror2, CAM-1, also has the kinase activity-dependent and -independent functions (9, 12, 13). Furthermore, CAM-1 exhibits the cytoplasmic region-independent functions, including cell migration (17), synaptic transmission at the neuromuscular junction (10), and inhibition of the β-catenin-dependent signaling pathway (11), although their underlying molecular mechanisms remain to be determined. However, it is unknown whether or not Ror2 also exhibits the cytoplasmic region-independent functions in other organisms.Dishevelled (Dvl) is an essential mediator of both the β-catenin-dependent and -independent signaling pathways. We have previously reported that both Ror2 and Dvl are required for Wnt5a-induced cell migration (28). However, the relationship between Ror2 and Dvl in Wnt5a signaling remains unclear. It has been reported that Dvl has an ability to form dynamic polymers, which are crucial for activating the β-catenin-dependent signaling pathway probably by serving as a scaffold for Axin recruitment (39, 41). However, there is no direct evidence showing that Wnt stimulation indeed induces dynamic formation of Dvl polymers. In addition, it remains unclear whether or not the polymerization of Dvl is involved in the β-catenin-independent signaling pathway.In the present study we show that Wnt5a induces dynamic polymerization of Dvl2 via a receptor complex containing both Ror2 and Frizzled (Fz)7, even in the absence of the cytoplasmic region of Ror2. We further provide evidence indicating that Ror2/Fz7 receptor complex plays an important role in Wnt5a/Rac1/AP-1 pathway by regulating polymerization of Dvl2.     

Functional Consequences of Wnt-induced Dishevelled 2 Phosphorylation in Canonical and Noncanonical Wnt Signaling

Dishevelled (Dvl) proteins are intracellular effectors of Wnt signaling that have essential roles in both canonical and noncanonical Wnt pathways. It has long been known that Wnts stimulate Dvl phosphorylation, but relatively little is known about its functional significance. We have previously reported that both Wnt3a and Wnt5a induce Dvl2 phosphorylation that is associated with an electrophoretic mobility shift and loss of recognition by monoclonal antibody 10B5. In the present study, we mapped the 10B5 epitope to a 16-amino acid segment of human Dvl2 (residues 594–609) that contains four Ser/Thr residues. Alanine substitution of these residues (P4m) eliminated the mobility shift induced by either Wnt3a or Wnt5a. The Dvl2 P4m mutant showed a modest increase in canonical Wnt/β-catenin signaling activity relative to wild type. Consistent with this finding, Dvl2 4Pm preferentially localized to cytoplasmic puncta. In contrast to wild-type Dvl2, however, the P4m mutant was unable to rescue Wnt3a-dependent neurite outgrowth in TC-32 cells following suppression of endogenous Dvl2/3. Earlier work has implicated casein kinase 1δ/ϵ as responsible for the Dvl mobility shift, and a CK1δ in vitro kinase assay confirmed that Ser⁵⁹⁴, Thr⁵⁹⁵, and Ser⁵⁹⁷ of Dvl2 are CK1 targets. Alanine substitution of these three residues was sufficient to abrogate the Wnt-dependent mobility shift. Thus, we have identified a cluster of Ser/Thr residues in the C-terminal domain of Dvl2 that are Wnt-induced phosphorylation (WIP) sites. Our results indicate that phosphorylation at the WIP sites reduces Dvl accumulation in puncta and attenuates β-catenin signaling, whereas it enables noncanonical signaling that is required for neurite outgrowth.     

Regulation of Phosphatidylinositol Kinases and Metabolism by Wnt3a and Dvl

Wnt signaling plays important roles in various physiological and pathophysiological processes. The pathway that leads to β-catenin stabilization is initiated by Wnt binding to its cell surface receptors, which induces the formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) via activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) type I. Here, we show that Wnt also stimulated the production of phosphatidylinositol 4-phosphate (PtdIns(4)P), which depended on Frizzled (Fz), Dishevelled (Dvl), and phosphatidylinositol 4-kinase (PI4K) type IIα in HEK293T cells. Dvl directly interacted with and activated PI4KIIα by increasing its V_max for ATP and PtdIns. In addition, Dvl regulated PI4KIIα and PIP5KI via different domains. Moreover, Dvl, PI4KIIα, and PIP5KI appeared to form a ternary complex upon Wnt3a stimulation. This complex may allow efficient production of PtdIns(4,5)P₂ from PtdIns, which is far more abundant than PtdIns(4)P in cells. Therefore, this study provides new insights into the mechanism by which Wnt3a regulates the production of PtdIns(4,5)P₂.The Wnt family of secretory glycoproteins plays important roles in regulation of embryonic development and tumorigenesis. They also regulate many other physiological and pathophysiological processes, including bone development, neuronogenesis, adipogenesis, myogenesis, organogenesis, and lipid and glucose metabolism (1–5). Studies using Drosophila and Xenopus embryos as well as mammalian cells have established a canonical Wnt signaling pathway that leads to stabilization of β-catenin. In the absence of Wnt, a number of proteins, including Axin, adenomatous polyposis coli (APC), casein kinase 1 (CK1), glycogen synthase kinase-3β (GSK3β),³ form a complex that facilitates β-catenin phosphorylation by CK1 and GSK3β. This phosphorylation targets β-catenin for ubiquitination and proteasome-mediated proteolytic degradation (3, 6). Some of the Wnt proteins bind to two cell surface receptors Fz and low density lipoprotein receptor-related protein (LRP) 5/6 and initiate a signaling cascade that eventually leads to the suppression of β-catenin phosphorylation by GSK3β and stabilization of β-catenin.Because the finding that the canonical Wnt proteins transduce signals by inducing the interaction between LRP5/6 and Axin (7), more has been learned about the mechanisms by which this interaction is regulated by Wnt proteins. Studies have indicated that two phosphorylation events at the C-terminal intracellular domain of LRP5/6, the phosphorylation of Thr¹⁴⁷⁹ by CKIγ (8, 9) and of Ser¹⁴⁹⁰ by GSK3 (10, 11), were required for the interaction. We recently showed that Wnt3a stimulated the production of PtdIns (4,5)P₂, which in turn regulated the phosphorylation of LRP5/6 at Thr¹⁴⁷⁹ and Ser¹⁴⁹⁰ (12). We also showed that Wnt3a regulated phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) activity by inducing the interaction between Dvl and PIP5KI (12). Moreover, Dvl could directly stimulate the lipid kinase activity of PIP5KI (12).PtdIns(4,5)P₂ plays important roles in various cellular functions, including membrane trafficking, cytoskeletal reorganization, migration, ion channel activation, and signal transduction (13). It, however, represents less than 1% of plasma membrane phospholipids and is primarily synthesized in most cells by sequential phosphorylation of PtdIns on the D4 and D5 positions of the inositol ring by two PtdIns kinases, PI4K and PIP5KI, respectively (14, 15). While PtdIns(4)P, the substrate for PIP5KI, is also accounted for around 1% of plasma membrane phospholipids, PtdIns, the substrate for PI4K, is very abundant. Thus, Wnt3a may have to stimulate PI4K activity to provide enough substrate for PIP5KI in PtdIns(4,5)P₂ production.Two types of PI4K (PI4KI and PI4KII) have been characterized in mammalian cells. There are two isoforms of PI4KII (PI4KIIα and PI4KIIβ) and two isoforms of PI4KI (PI4KIα and PI4KIβ) (16). In our previous study, we demonstrated the involvement of PI4KIIα in Wnt signaling. siRNA-mediated knockdown in mammalian cells and morpholino-mediated suppression in Xenopus embryos of PI4KIIα inhibited LRP6 phosphorylation and Wnt signaling. In this report, we examined whether Wnt3a regulates the lipid kinase activity of PI4KIIα and found that Wnt3a could induce an increase in the level of PtdIns(4)P in a Dvl- and Fz-dependent manner. In addition, the Dvl protein was found to directly interact with and activate PI4KIIα. Moreover, different domains of Dvl appeared to be involved in the regulation of PI4KIIα and PIP5KI, and Wnt3a induced the formation of a complex of Dvl, PI4KIIα, and PIP5KI possibly for more efficient production of PtdIns (4,5)P₂ in cells.     

The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells

Introduction

The goals of this study were to examine the oxemic regulation of Wnt signaling to explore whether Wnt signaling accelerates the age-related degeneration of nucleus pulposus cells, and if so, to define the mechanism underlying this effect. We investigated the expression of Klotho, a newly identified antiaging gene, and whether its regulation is attributable to the suppression of Wnt signaling.

Methods

Rat nucleus pulposus cells were cultured under normoxic (21% O₂) or hypoxic (2% O₂) conditions, and the expression and promoter activity of Wnt signaling and Klotho were evaluated. The effect of Klotho protein was examined with transfection experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, senescence-associated β-galactosidase staining, and cell-cycle analysis. To determine the methylation status of the Klotho promoter region, bisulfite genomic sequencing analysis was performed. Its relation with the activation of Wnt signaling was assessed. We also examined whether the expression of Klotho could block the effects of pathological Wnt expression in nucleus pulposus cells.

Results

Nucleus pulposus cells exhibited increased β-catenin mRNA and protein under the hypoxic condition. Klotho protein was expressed in vivo, and protein and messenger RNA expression decreased under the hypoxic condition. Klotho treatment decreased cell proliferation and induced the quiescence of nucleus pulposus cells. In addition, Klotho treatment inhibited expression of β-catenin gene and protein compared with untreated control cells.

Conclusions

These data indicate that Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells. These results suggest that the expression of Klotho is regulated by the balance between upregulation and downregulation of Wnt signaling.     

Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust Senescence Associated Heterochromatin Foci

Background

Cellular senescence is a permanent growth arrest that occurs in response to cellular stressors, such as telomere shortening or activation of oncogenes. Although the process of senescence growth arrest is somewhat conserved between mouse and human cells, there are some critical differences in the molecular pathways of senescence between these two species. Recent studies in human fibroblasts have defined a cell signaling pathway that is initiated by repression of a specific Wnt ligand, Wnt2. This, in turn, activates a histone chaperone HIRA, and culminates in formation of specialized punctate domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF), that are enriched in the histone variant, macroH2A. SAHF are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. We asked whether this Wnt2-HIRA-SAHF pathway is conserved in mouse fibroblasts.

Results

We show that mouse embryo fibroblasts (MEFs) and mouse skin fibroblasts, do not form robust punctate SAHF in response to an activated Ras oncogene or shortened telomeres. However, senescent MEFs do exhibit elevated levels of macroH2A staining throughout the nucleus as a whole. Consistent with their failure to fully activate the SAHF assembly pathway, the Wnt2-HIRA signaling axis is not overtly regulated between proliferating and senescent mouse cells.

Conclusions

In addition to the previously defined differences between mouse and human cells in the mechanisms and phenotypes associated with senescence, we conclude that senescent mouse and human fibroblasts also differ at the level of chromatin and the signaling pathways used to regulate chromatin. These differences between human and mouse senescence may contribute to the increased propensity of mouse fibroblasts (and perhaps other mouse cell types) to become immortalized and transformed, compared to human cells.     

NEDD4L regulates convergent extension movements in Xenopus embryos via Disheveled-mediated non-canonical Wnt signaling

During the early vertebrate body plan formation, convergent extension (CE) of dorsal mesoderm and neurectoderm is coordinated by the evolutionarily conserved non-canonical Wnt/PCP signaling. Disheveled (Dvl), a key mediator of Wnt/PCP signaling, is essential for the medial–lateral polarity formation in the cells undergoing convergent extension movements. NEDD4L, a highly conserved HECT type E3 ligase, has been reported to regulate the stability of multiple substrates including Dvl2. Here we demonstrate that NEDD4L is required for the cellular polarity formation and convergent extension in the early Xenopus embryos. Depletion of NEDD4L in early Xenopus embryos results in the loss of mediolateral polarity of the convergent-extending mesoderm cells and the shortened body axis, resembling those defects caused by the disruption of non-canonical Wnt signaling. Depletion of xNEDD4L also blocks the elongation of the animal explants in response to endogenous mesoderm inducing signals and partially compromises the expression of Brachyury. Importantly, reducing Dvl2 expression can largely rescue the cellular polarity and convergent extension defects in NEDD4L-depleted embryos and explants. Together with the data that NEDD4L reduces Dvl2 protein expression in the frog embryos, our findings suggest that regulation of Dvl protein levels by NEDD4L is essential for convergent extension during early Xenopus embryogenesis.     

SIRT1 inhibits adipogenesis and promotes myogenic differentiation in C3H10T1/2 pluripotent cells by regulating Wnt signaling

Background

The directed differentiation of mesenchymal stem cells (MSCs) is tightly controlled by a complex network. Wnt signaling pathways have an important function in controlling the fate of MSCs. However, the mechanism through which Wnt/β-catenin signaling is regulated in differentiation of MSCs remains unknown. SIRT1 plays an important role in the regulation of MSCs differentiation.

Results

This study aimed to determine the effect of sirtuin 1 (SIRT1) on adipogenesis and myogenic differentiation of C3H10T1/2 cells. First, the MSC commitment and differentiation model was established by using 5-azacytidine. Using the established model, C3H10T1/2 cells were treated with SIRT1 activator/inhibitor during differentiation. The results showed that resveratrol inhibits adipogenic differentiation and improves myogenic differentiation, whereas nicotinamide promotes adipogenic differentiation. Notably, during commitment, resveratrol blocked adipocyte formation and promoted myotubes differentiation, whereas nicotinamide enhanced adipogenic potential of C3H10T1/2 cells. Furthermore, resveratrol elevated the expression of Cyclin D1 and β-catenin in the early stages. The luciferase assay showed that knockdown SIRT1 inhibits Wnt/β-catenin signaling, while resveratrol treatment or overexpression SIRT1 activates Wnt/β-catenin signaling. SIRT1 suppressed the expression of Wnt signaling antagonists sFRP2 and DACT1. Knockdown SIRT1 promoted adipogenic potential of C3H10T1/2 cells, whereas overexpression SIRT1 inhibited adipogenic differentiation and promoted myogenic differentiation.

Conclusions

Together, our results suggested that SIRT1 inhibits adipogenesis and stimulates myogenic differentiation by activating Wnt signaling.

     

Selective Modulation of Wnt Ligands and Their Receptors in Adipose Tissue by Chronic Hyperadiponectinemia

Background

Adiponectin-transgenic mice had many small adipocytes in both subcutaneous and visceral adipose tissues, and showed higher sensitivity to insulin, longer life span, and reduced chronic inflammation. We hypothesized that adiponectin regulates Wnt signaling in adipocytes and thereby modulates adipocyte proliferation and chronic inflammation in adipose tissue.

Materials and Methods

We examined the expression of all Wnt ligands and their receptors and the activity of Wnt signaling pathways in visceral adipose tissue from wild-type mice and two lines of adiponectin-transgenic mice. The effects of adiponectin were also investigated in cultured 3T3-L1 cells.

Results

The Wnt5b, Wnt6, Frizzled 6 (Fzd6), and Fzd9 genes were up-regulated in both lines of transgenic mice, whereas Wnt1, Wnt2, Wnt5a, Wnt9b, Wnt10b, Wnt11, Fzd1, Fzd2, Fzd4, Fzd7, and the Fzd coreceptor low-density-lipoprotein receptor-related protein 6 (Lrp6) were reduced. There was no difference in total β-catenin levels in whole-cell extracts, non-phospho-β-catenin levels in nuclear extracts, or mRNA levels of β-catenin target genes, indicating that hyperadiponectinemia did not affect canonical Wnt signaling. In contrast, phosphorylated calcium/calmodulin-dependent kinase II (p-CaMKII) and phosphorylated Jun N-terminal kinase (p-JNK) were markedly reduced in adipose tissue from the transgenic mice. The adipose tissue of the transgenic mice consisted of many small cells and had increased expression of adiponectin, whereas cyclooxygenase-2 expression was reduced. Wnt5b expression was elevated in preadipocytes of the transgenic mice and decreased in diet-induced obese mice, suggesting a role in adipocyte differentiation. Some Wnt genes, Fzd genes, and p-CaMKII protein were down-regulated in 3T3-L1 cells cultured with a high concentration of adiponectin.

Conclusion

Chronic hyperadiponectinemia selectively modulated the expression of Wnt ligands, Fzd receptors and LRP coreceptors accompanied by the inhibition of the Wnt/Ca²⁺ and JNK signaling pathways, which may be involved in the altered adipocyte cellularity, endogenous adiponectin production, and anti-inflammatory action induced by hyperadiponectinemia.