PDGF promotes the proliferation of human melanoblasts and differentiation of melanocytes 104, indicating that adipose secreted PDGF may also regulate McSCs activation and differentiation.

Mechanistically, knockout of Kindlin-1 promotes cutaneous epithelial stem cells differentiation via inhibiting alpha (v) beta (6) integrin mediated TGF-beta1 liberation and promoting integrin independent Wnt ligand expression to activate Wnt and beta-catenin signaling 82.

Injecting Wnt inhibitor DKK1 into skin inhibits TPA induced the proliferation and differentiation of McSCs 52.

Injecting Wnt inhibitor DKK1 into skin inhibits TPA induced the proliferation and differentiation of McSCs 52.

So the white color can be due to the absence of melanocytes by non migration or death of melanocytes, or due to the suppression of differentiation by agouti or other inhibitors.

HGF, SCF, and End3 have been revealed to promote melanoblast or melanocyte proliferation and differentiation XREF_BIBR, XREF_BIBR.

HGF, SCF, and End3 have been revealed to promote melanoblast or melanocyte proliferation and differentiation XREF_BIBR, XREF_BIBR.

In contrast, blockade of CD16 produced no inhibitory effect on CRP-induced expression of CXCL8, CCL2, MMP9 and IL-6 by RA-FLSs ( xref ), suggesting that CRP may not signal through the CD16 to induce joint inflammation in vitro .

However, blocking CD32 but not CD64 to inhibit CRP induced FLS proliferation, invasiveness, and proinflammatory cytokine CXCL8 production revealed a major role for CD32 signaling in synovial inflammation, although CRP via CD64, not CD32, to induce MMP9 expression was noticed.

Interestingly, CRP-induced MMP9 expression and invasion on RA-FLSs were p38-dependent as addition of a p38 inhibitor (SB202190) but not a NF-κB inhibitor (PDTC) was capable of inhibiting CRP-induced MMP9 expression and cell invasion.

In contrast, blockade of CD16 produced no inhibitory effect on CRP-induced expression of CXCL8, CCL2, MMP9 and IL-6 by RA-FLSs ( xref ), suggesting that CRP may not signal through the CD16 to induce joint inflammation in vitro .

However, CRP- induced expression of CXCL8 was CD32-dependent as it was blunted by the antibody against CD32, whereas CRP-induced MMP9 was blocked by the antibody to CD64, demonstrating that differential signaling mechanisms for CRP in regulating CXCL8 and MMP9 expression in RA-FLSs.

In contrast, blockade of CD16 produced no inhibitory effect on CRP-induced expression of CXCL8, CCL2, MMP9 and IL-6 by RA-FLSs ( xref ), suggesting that CRP may not signal through the CD16 to induce joint inflammation in vitro .

As shown in xref , CRP-induced expression of CCL2 and IL-6 was blocked by either neutralizing antibody to CD32 or CD64 or both, suggesting that CRP signals through both CD32/CD64 to induce expression of CCL2 and IL-6.

As shown in XREF_FIG, CRP induced expression of CCL2 and IL-6 was blocked by either neutralizing antibody to CD32 or CD64 or both, suggesting that CRP signals through both CD32 and CD64 to induce expression of CCL2 and IL-6.

In contrast, blockade of CD16 produced no inhibitory effect on CRP-induced expression of CXCL8, CCL2, MMP9 and IL-6 by RA-FLSs ( xref ), suggesting that CRP may not signal through the CD16 to induce joint inflammation in vitro .

As shown in xref , CRP-induced expression of CCL2 and IL-6 was blocked by either neutralizing antibody to CD32 or CD64 or both, suggesting that CRP signals through both CD32/CD64 to induce expression of CCL2 and IL-6.

As shown in XREF_FIG, CRP induced expression of CCL2 and IL-6 was blocked by either neutralizing antibody to CD32 or CD64 or both, suggesting that CRP signals through both CD32 and CD64 to induce expression of CCL2 and IL-6.

We thus blocked CD32 or CD64 or both with neutralizing antibodies to differentially determine the signaling mechanisms of CRP-induced cytokine expression.

It has been reported that CRP-induced cytokine expression is also regulated by TWIST transcriptionally in myeloma cells ( xref ).

Thus, it is highly possible that high concentration of CRP in synovial fluid in patients with RA may directly bind primarily CD32 to activate p38 MAP kinase and NF-kappaB signaling in RA-FLSs to differentially regulate synovial inflammation.

In vitro studies confirmed this notion and found that CRP was able to upregulate both CD32 and CD64 and induced FLS proliferation, invasion, and pro inflammatory expression by increasing production of CCL2, CXCL8, IL-6, MMP2, MMP9 while suppressing an anti-inflammatory cytokine IL-10 expression.

RA-FLS was a major cell type responsible for CRP production in RA patients, accounting for more than 65% of CRP producing cells as identified by co-expressing CRP and vimentin in the inflamed synovial tissues in patients with RA.

As shown in XREF_FIG, multiplex cytokine assay kits assays showed that addition of CRP dose-dependently upregulated CCL2, CXCL8, IL-6, MMP2, MMP9 in RA-FLS but not in HFLS, although expression of IL-1beta and TNFalpha was not significantly changed (XREF_FIG).

In vitro studies confirmed this notion and found that CRP was able to upregulate both CD32 and CD64 and induced FLS proliferation, invasion, and pro inflammatory expression by increasing production of CCL2, CXCL8, IL-6, MMP2, MMP9 while suppressing an anti-inflammatory cytokine IL-10 expression.

In vitro studies confirmed this notion and found that CRP was able to upregulate both CD32 and CD64 and induced FLS proliferation, invasion, and pro inflammatory expression by increasing production of CCL2, CXCL8, IL-6, MMP2, MMP9 while suppressing an anti-inflammatory cytokine IL-10 expression.

As shown in XREF_FIG, multiplex cytokine assay kits assays showed that addition of CRP dose-dependently upregulated CCL2, CXCL8, IL-6, MMP2, MMP9 in RA-FLS but not in HFLS, although expression of IL-1beta and TNFalpha was not significantly changed (XREF_FIG).

In vitro studies confirmed this notion and found that CRP was able to upregulate both CD32 and CD64 and induced FLS proliferation, invasion, and pro inflammatory expression by increasing production of CCL2, CXCL8, IL-6, MMP2, MMP9 while suppressing an anti-inflammatory cytokine IL-10 expression.

As shown in XREF_FIG, multiplex cytokine assay kits assays showed that addition of CRP dose-dependently upregulated CCL2, CXCL8, IL-6, MMP2, MMP9 in RA-FLS but not in HFLS, although expression of IL-1beta and TNFalpha was not significantly changed (XREF_FIG).

In vitro studies confirmed this notion and found that CRP was able to upregulate both CD32 and CD64 and induced FLS proliferation, invasion, and pro inflammatory expression by increasing production of CCL2, CXCL8, IL-6, MMP2, MMP9 while suppressing an anti-inflammatory cytokine IL-10 expression.

As shown in XREF_FIG, multiplex cytokine assay kits assays showed that addition of CRP dose-dependently upregulated CCL2, CXCL8, IL-6, MMP2, MMP9 in RA-FLS but not in HFLS, although expression of IL-1beta and TNFalpha was not significantly changed (XREF_FIG).

In vitro studies confirmed this notion and found that CRP was able to upregulate both CD32 and CD64 and induced FLS proliferation, invasion, and pro inflammatory expression by increasing production of CCL2, CXCL8, IL-6, MMP2, MMP9 while suppressing an anti-inflammatory cytokine IL-10 expression.

As shown in XREF_FIG, multiplex cytokine assay kits assays showed that addition of CRP dose-dependently upregulated CCL2, CXCL8, IL-6, MMP2, MMP9 in RA-FLS but not in HFLS, although expression of IL-1beta and TNFalpha was not significantly changed (XREF_FIG).

In vitro studies confirmed this notion and found that CRP was able to upregulate both CD32 and CD64 and induced FLS proliferation, invasion, and pro inflammatory expression by increasing production of CCL2, CXCL8, IL-6, MMP2, MMP9 while suppressing an anti-inflammatory cytokine IL-10 expression.

This was further confirmed by the ability of pre-treating RA-FLS with a NF-kappaB inhibitor, PDTC (100 mumol/L) to inhibit CRP induced proliferation (XREF_FIG) and upregulation of CXCL8, CCL2.

Here we tested the hypothesis that CRP may be produced locally by FLSs and functions to induce the synovial inflammation in patients with RA.

CRP may promote synovial inflammation via mechanism associated with activation of CD32/64- p38 and NF-kappaB signaling.

In the present study, we found that CRP signaled primarily through CD32, to a less extent of CD64, to differentially regulate joint inflammation.

CRP Promotes RA-FLS Pro inflammatory Response Differentially via the CD32/64-p 38 and NF-kappaB-Dependent Mechanisms in vitro.

CRP can induce synovial inflammation via mechanisms associated with activation of CD32/64-p 38 and NF-kappaB signaling.

This was supported by the findings that CRP induced activation of p38 MAP kinase and NF-kappaB signaling was blunted by neutralizing antibodies against CD32 but not CD64.

To examine whether CRP induces NF-kappaB nuclear translation, immunofluorescence and subcellular fractionation were performed.

We found that p-Y42 RhoA also readily translocated to the nucleus, similarly to β-catenin, upon Wnt3A signaling ( xref A).

However, the mechanism by which p -Tyr42 RhoA translocates to the nucleus remains elusive.

We found that p-Y42 RhoA also readily translocated to the nucleus, similarly to beta-catenin, upon Wnt3A signaling (XREF_FIG A).

However, the mechanism by which p-Tyr42 RhoA translocates to the nucleus remains elusive.

In absence of Wnt signaling, glycogen synthase kinase-3 beta (GSK-3beta) phosphorylates beta-catenin in a ' destruction complex '.

In presence of Wnt, there is the stimulation of cells in the canonical pathway as GSK-3beta phosphorylates lipoprotein receptor related protein 6 (LRP6) rather than beta-catenin, which is necessary for Axin to bind to LRP6 together with GSK-3beta and APC [XREF_BIBR].

Consequently, GSK-3beta can not interact with and phosphorylate beta-catenin, resulting in increased beta-catenin stability.

In particular, Tyr42 of RhoA phosphorylated by c-Src binds to IKKgamma, leading to IKKbeta activation, IkappaB phosphorylation and its degradation, resulting in NF-kappaB activation and tumorigenesis [XREF_BIBR].

These results suggest that upon Wnt3A stimulation, p-Tyr216 GSK-3beta and dephospho-Ser9 GSK-3beta, both of which are known to be active forms of GSK-3beta, are critical for beta-catenin accumulation along with Tyr416 phosphorylation of Src and Tyr42 phosphorylation of RhoA (XREF_FIG C).

In a previous report, we found that Tyr42 phosphorylation of RhoA (p-Tyr42 RhoA) is induced by hydrogen peroxide (H 2 O 2) in RAW264.7 cells, a macrophage cell line [XREF_BIBR].

Here, hydrogen peroxide also induced Tyr42 phosphorylation of Rho GTPase in HEK293T cells (XREF_FIG A).

Likewise, si-RhoA also prevented beta-catenin activity, and reconstitution of RhoA WT and phospho-mimetic Y42E restored the beta-catenin activity, but not with dephospho-mimetic RhoA Y42F, it having a remarkably reduced beta-catenin activity (XREF_FIG F).

However, in consistent to the previous report [XREF_BIBR], we revealed that si-RhoA impaired beta-catenin accumulation and reconstitution of RhoA restored beta-catenin accumulation (XREF_FIG A), suggesting that RhoA activity and beta-catenin stability are very closely linked, although the underlying mechanism by which RhoA induces beta-catenin accumulation in response to Wnt3A remains to be revealed.

Conversely, RhoA can be inactivated by GTPase activating proteins (GAPs) which catalyze GTP hydrolysis, leading to a GDP bound RhoA, which is inactive.

RhoA WT and RhoA Y42E transfection to cells also had increased expression of cyclin D1 and c-Myc in presence of Wnt3A, whereas RhoA Y42F transfection inhibited this cyclin D1 and c-Myc up-regulation (XREF_FIG F).

RhoA WT and RhoA Y42E transfection to cells also had increased expression of cyclin D1 and c-Myc in presence of Wnt3A, whereas RhoA Y42F transfection inhibited this cyclin D1 and c-Myc up-regulation (XREF_FIG F).

In this study, we found that Wnt3A induces interaction of p-Tyr42 RhoA and beta-catenin and p-Tyr42 RhoA delivers beta-catenin to the nucleus, where p-Tyr42 RhoA as well as beta-catenin regulates expression of specific genes such as Vim by binding to Vim promoter.

We next tried to identify whether GDP/GTP bound states of RhoA determine the interaction between RhoA and beta-catenin.

In addition, it was demonstrated that p-Tyr42 RhoA directly interact with beta-catenin and nuclear localization of p-Tyr42 RhoA is required for nuclear delivery of beta-catenin.

We next tried to identify whether GDP/GTP-bound states of RhoA determine the interaction between RhoA and β-catenin.

Recombinant GST-β-catenin binding to RhoA-GTPγS or RhoA-GDP in vitro was not significantly different, suggesting that GDP or GTP binding to RhoA is not critical for regulating interaction between RhoA with β-catenin ( xref D).

To identify the specific domain of β-catenin associating with RhoA, we expressed and purified GST-fusions of β-catenin domains composed of amino acids (aa) 1–140, 141–390, 391–662 and 663–782 ( xref E and F).

Similarly, GST-RhoA phosphorylated by Src and ATP [ xref ] also readily bound to β-catenin in vitro , irrespective of GDP- or GTPγS-preloaded RhoA, suggesting that phosphorylation of Tyr42, but not GTP/GDP-binding state is critical for interaction between RhoA and β-catenin ( xref D).

The 3D of RhoA revealed Tyr42 residue positioning in extended β2 region did not dramatically alter 3D location, while Tyr34 in switch 1 (aa 28–38) and Tyr66 in switch 2 (aa 61–78) regions revealed significant alteration [ xref ] ( xref E), suggesting that GDP or GTP may not contribute p -Tyr42 RhoA binding to β-catenin.

Interaction of p -Tyr42 RhoA with β-catenin.

Also, RhoA preferentially binds to N -terminal domain (NTD: aa 1–140) of β-catenin ( xref G).

It is remarkable that RhoA Y42F (dephospho-mimetic) did not interfere with β-catenin accumulation ( xref A), but RhoA Y42F could not bind to β-catenin ( xref B and 5C).

Since expression of vimentin can be also induced by β-catenin [ xref ], we speculate p -Tyr42 RhoA binding to β-catenin facilitate vimentin expression.

Meanwhile, p -Tyr42 RhoA bound to β-catenin via the N -terminal domain of β-catenin, thereby leading to the nuclear translocation of p -Tyr42 RhoA/β-catenin complex.

In this study, we found that Wnt3A induces interaction of p -Tyr42 RhoA and β-catenin and p -Tyr42 RhoA delivers β-catenin to the nucleus, where p -Tyr42 RhoA as well as β-catenin regulates expression of specific genes such as Vim by binding to Vim promoter.

In addition, it was demonstrated that p -Tyr42 RhoA directly interact with β-catenin and nuclear localization of p -Tyr42 RhoA is required for nuclear delivery of β-catenin.

Of note, beta-catenin binds to Lef1 and activates Lef1 transcription complex, leading to down-regulation of E-cadherin expression [XREF_BIBR].

Of note, β-catenin binds to Lef1 and activates Lef1 transcription complex, leading to down-regulation of E -cadherin expression [ xref ].

p -Tyr42 Rho binds to promoters of specific genes such as Vim.

In this study, as a novel mechanism, p -Tyr42 RhoA binds to promoter of Vim and regulates vimentin expression, suggesting that p -Tyr42 RhoA regulates EMT ( xref ).

Notably, p -Tyr42 RhoA as well as β-catenin was associated with the promoter of Vim , leading to increased expression of vimentin.

ROCK2 and p-Tyr42 RhoA bind to Src, where they transmit other signaling pathway through p-p47phox (XREF_FIG H, XREF_FIG and 3L).

Notably, p-Tyr42 RhoA preferentially bound to ROCK2, which in turn preferentially bound to p47phox, leading Ser345 phosphorylation of p47phox (XREF_FIG H and XREF_FIG).

Notably, p -Tyr42 RhoA preferentially bound to ROCK2, which in turn preferentially bound to p47phox, leading Ser345 phosphorylation of p47phox ( xref H and xref ).

It is notable that IKKgamma (also known as NEMO) facilitates RhoA activation via IKKgamma (NEMO) causing the dissociation of the RhoA and RhoGDI complex [XREF_BIBR].

It is notable that IKKγ (also known as NEMO) facilitates RhoA activation via IKKγ (NEMO) causing the dissociation of the RhoA-RhoGDI complex [ xref ].

GST-RhoA WT, Y42E and Y42F preloaded with GDP or GTPγS were incubated recombinant purified β-catenin in vitro .

Similarly, GST-RhoA phosphorylated by Src and ATP [ xref ] also readily bound to β-catenin in vitro , irrespective of GDP- or GTPγS-preloaded RhoA, suggesting that phosphorylation of Tyr42, but not GTP/GDP-binding state is critical for interaction between RhoA and β-catenin ( xref D).

Recombinant GST-β-catenin binding to RhoA-GTPγS or RhoA-GDP in vitro was not significantly different, suggesting that GDP or GTP binding to RhoA is not critical for regulating interaction between RhoA with β-catenin ( xref D).

RhoA preloaded with GTPγS readily bound to aa 1–140 NTD of GST-β-catenin domain, conjugated to beads, whereas other domain fusion proteins only marginally bound to RhoA ( xref G).

Conversely, RhoA can be inactivated by GTPase activating proteins (GAPs) which catalyze GTP hydrolysis, leading to a GDP bound RhoA, which is inactive.

As a small GTPase, RhoA can be activated by several guanine nucleotide exchange factors (GEFs) resulting in GTP binding to RhoA, representing an activated RhoA.

Remarkably, si-RhoA impaired beta-catenin accumulation, but re-constitutive dephospho-mimetic RhoA Y42F as well as RhoA WT and phospho-mimetic RhoA Y42E allowed recovery of beta-catenin accumulation (XREF_FIG A).

However, in consistent to the previous report [XREF_BIBR], we revealed that si-RhoA impaired beta-catenin accumulation and reconstitution of RhoA restored beta-catenin accumulation (XREF_FIG A), suggesting that RhoA activity and beta-catenin stability are very closely linked, although the underlying mechanism by which RhoA induces beta-catenin accumulation in response to Wnt3A remains to be revealed.

One clue for this function is that the RhoA effector protein, ROCK2, which was supposed to be activated by p-Tyr42 RhoA (XREF_FIG H), may enhance the activation of p300 acetyltransferase by phosphorylation [XREF_BIBR].

The results suggest that ROCK2 is a preferential downstream effector protein of p-Tyr42 RhoA while ROCK1 is activated dominantly by RhoA (XREF_FIG J).

The results suggest that ROCK2 is a preferential downstream effector protein of p -Tyr42 RhoA while ROCK1 is activated dominantly by RhoA ( xref J).

Consistent to the result of XREF_FIG B, si-RhoA abolished superoxide production, and reconstituted RhoA WT and phospho-mimetic RhoA Y42E restored superoxide production.

Of note, β-catenin binds to Lef1 and activates Lef1 transcription complex, leading to down-regulation of E -cadherin expression [ xref ].

3.4 Wnt3A induces formation of RhoA, beta, and catenin complex.

Here, we found that Wnt3A also induces the complex formation of beta-catenin and RhoA, particularly p-Y42 RhoA.

Wnt3A induced nuclear localization of beta-catenin (XREF_FIG A).

Here, we found that Wnt3A also induces the complex formation of beta-catenin and RhoA, particularly p-Y42 RhoA.

Of note, vimentin can activate RhoA through GEF-H1 (also known as ARHGEF2) [ xref ].

Of note, vimentin can activate RhoA through GEF-H1 (also known as ARHGEF2) [XREF_BIBR].

ROS scavenger, NAC and NADPH oxidase inhibitors, DPI and apocynin abolished upregulation of p-Tyr42 Rho, beta-catenin, p-Tyr416 Src, p-Ser9 GSK-3beta and p-Tyr216 GSK-3beta upon Wnt3A in HEK293T cells, suggesting that superoxide is closely involved in the increases of the above molecules via Wnt3A (XREF_FIG C).

ROS scavenger, NAC and NADPH oxidase inhibitors, DPI and apocynin abolished upregulation of p-Tyr42 Rho, beta-catenin, p-Tyr416 Src, p-Ser9 GSK-3beta and p-Tyr216 GSK-3beta upon Wnt3A in HEK293T cells, suggesting that superoxide is closely involved in the increases of the above molecules via Wnt3A (XREF_FIG C).

As a small GTPase, RhoA can be activated by several guanine nucleotide exchange factors (GEFs) resulting in GTP binding to RhoA, representing an activated RhoA.

Thereby, it is possible that p -Tyr42 RhoA GTPase causes the acetylation of β-catenin through p300 acetyltransferase, remarkably enhancing β-catenin activity as a co-activator of TCF4 in the nucleus.

Thereby, it is possible that p-Tyr42 RhoA GTPase causes the acetylation of beta-catenin through p300 acetyltransferase, remarkably enhancing beta-catenin activity as a co-activator of TCF4 in the nucleus.

RhoA GTPase phosphorylated at tyrosine 42 by src kinase binds to β-catenin and contributes transcriptional regulation of vimentin upon Wnt3A.

RhoA GTPase phosphorylated at tyrosine 42 by src kinase binds to beta-catenin and contributes transcriptional regulation of vimentin upon Wnt3A.

Meanwhile, p-Tyr42 RhoA bound to β-catenin via the N-terminal domain of β-catenin, thereby leading to the nuclear translocation of p-Tyr42 RhoA/β-catenin complex.

Meanwhile, p-Tyr42 RhoA bound to beta-catenin via the N-terminal domain of beta-catenin, thereby leading to the nuclear translocation of p-Tyr42 RhoA, beta, and catenin complex.

Notably, p-Tyr42 RhoA as well as β-catenin was associated with the promoter of Vim, leading to increased expression of vimentin.

RhoA GTPase phosphorylated at tyrosine 42 by src kinase binds to beta-catenin and contributes transcriptional regulation of vimentin upon Wnt3A.

Wnt3A also induced accumulation of beta-catenin along with activations of RhoA and ROCK1.

KLK8 degrades VE-cadherin , thereby promoting plakoglobin nuclear translocation The KLK family is considered to cleave kininogens to release kinin 15 .

KLK8 degrades VE-cadherin, thereby promoting plakoglobin nuclear translocation.

As shown in supplemental xref , treatment of cardiac fibroblasts with Ad-KLK8 induced mRNA expression of the proliferation-related genes Ki67, proliferating cell nuclear antigen (PCNA) and cyclin D1 xref .

Protein expression levels of Ki67 and PCNA were also increased by KLK8 overexpression.

The HIF-1α inhibitor echinomycin not only inhibited basal TGF-β1 expression, but also blocked KLK8-induced TGF-β1 mRNA expression and release in HCAECs (Figure xref D).

However, ICG-001 treatment had no significant effect on KLK8-induced TGF-β1 expression ( xref F).

The present study found that upregulation of KLK8 leads to plakoglobin dependent nuclear translocation of p53, which binds to HIF-1alpha, and further enhances the transactivation effect of HIF-1alpha on the TGF-beta1 promoter and consequently upregulates TGF-beta1 expression at the transcriptional level.

As shown in supplemental xref , treatment of cardiac fibroblasts with Ad-KLK8 induced mRNA expression of the proliferation-related genes Ki67, proliferating cell nuclear antigen (PCNA) and cyclin D1 xref .

The HIF-1α inhibitor echinomycin not only inhibited basal TGF-β1 expression, but also blocked KLK8-induced TGF-β1 mRNA expression and release in HCAECs (Figure xref D).

A specificity protein-1 (Sp-1) consensus site was identified in the human KLK8 promoter and was found to mediate the high glucose induced KLK8 expression.

A specificity protein-1 (Sp-1) consensus site was identified in the human KLK8 promoter and was found to mediate the high glucose induced KLK8 expression.

It was found that the mRNA and protein expression levels of KLK8 were significantly upregulated in a dose dependent manner in high glucose treated HCAECs compared with those of normal glucose treated HCAECs, suggesting that high glucose stimulated KLK8 expression at the transcriptional level.

To explore the molecular mechanisms involved in the high glucose induced upregulation of KLK8 expression, HCAECs were treated with increasing concentration of glucose (15 and 25 mM) for 5 days.

The present study found that Ad-KLK8 infection resulted in a 2.88 ± 0.35 fold increase in the content of bradykinin in the culture medium of HCAECs.

In HCAECs, it was found that Ad-KLK8 increased the expression levels of α-SMA and vimentin, whereas it decreased the expression levels of CD31 and VE-cadherin, in a dose-dependent manner, suggesting that KLK8 overexpression is able to induce EndMT (Figure xref E-F).

In addition, the permeability of a confluent HCAEC monolayer was measured for 10-kDa FITC-dextran, and it was found that Ad-KLK8 treatment significantly increased endothelial permeability (Figure xref L).

Infection of HCAECs with increasing concentrations of KLK8 adenovirus (Ad-KLK8) led to an increase in KLK8 expression in a dose-dependent manner ( xref ).

As shown in supplemental xref , treatment of cardiac fibroblasts with Ad-KLK8 induced mRNA expression of the proliferation-related genes Ki67, proliferating cell nuclear antigen (PCNA) and cyclin D1 xref .

The present study then examined the composition of the endothelial cell culture medium after cell transfection with Ad-KLK8.

Adenovirus-mediated overexpression of KLK8 (Ad-KLK8) resulted in increases in endothelial cell damage, permeability and transforming growth factor (TGF)-β1 release in HCAECs.

The present study found that KLK8 overexpression significantly increased the association of p53 with Smad3, which was blocked by plakoglobin knockdown (Figure xref F).

KLK8 also induced the binding of p53 with Smad3, subsequently promoting pro-EndMT reprogramming via the TGF-β1/Smad signaling pathway in HCAECs.

KLK8 also induced the binding of p53 with Smad3, subsequently promoting pro EndMT reprogramming via the TGF-beta1 and Smad signaling pathway in HCAECs.

Previous study has found that plakoglobin interacts with p53 and increases its transcriptional activity, thereby regulating the expression of various genes involved in tumorigenesis and metastasis xref .

The association of plakoglobin with p53 in diabetic heart tissues was decreased in KLK8 -/- mice (Figure xref F).

As shown in Figure xref A, it was found that high glucose led to a significant increase in the association of plakoglobin with p53, which was blocked by KLK8 knockdown.

Diabetes mellitus increased the association of p53 with both HIF-1α and Smad3 in heart tissues, which was attenuated in KLK8 -/- mice (Figure xref F).

In addition, high glucose induced p53 binding to both HIF-1α and Smad3, which was blocked by siRNA targeting KLK8 and plakoglobin (Figure xref A-B).

The present study found that upregulation of KLK8 leads to plakoglobin-dependent nuclear translocation of p53, which binds to HIF-1α, and further enhances the transactivation effect of HIF-1α on the TGF-β1 promoter and consequently upregulates TGF-β1 expression at the transcriptional level.

Altogether, these data suggest that upregulation of KLK8 leads to plakoglobin-dependent association of p53 with HIF-1α, which further enhances the transactive effect of HIF-1α on the TGF-β1 promoter.

KLK8 overexpression induced the association of p53 with HIF-1α, which was blocked by plakoglobin knockdown (Figure xref F).

The present study found that upregulation of KLK8 leads to plakoglobin dependent nuclear translocation of p53, which binds to HIF-1alpha, and further enhances the transactivation effect of HIF-1alpha on the TGF-beta1 promoter and consequently upregulates TGF-beta1 expression at the transcriptional level.

Using ChIP analysis, it was found that high glucose treatment led to a significant increase in the binding of HIF-1α to the TGF-β1 promoter, which was largely blocked in the presence of KLK8 siRNA, plakoglobin siRNA (Figure xref C) or the p53 inhibitor pifithrin-α (Figure xref D).

Using ChIP analysis, the present study confirmed the binding of HIF-1α to the TGF-β1 promoter, which was significantly enhanced by KLK8 overexpression (Figure xref E).

KLK8 overexpression led to a significant increase in the association of plakoglobin with both p53 and TCF-4 (Figure xref C).

The present study found that plakoglobin was associated with p53 and TCF-4 in HCAECs (Figure xref C).

Notably, although KLK8 overexpression led to a significant increase in the association of plakoglobin with both p53 and TCF-4, KLK8-induced EndMT was reduced only by a p53 inhibitor, but not by the inhibitor of β-catenin/TCF-mediated transcription.

While cadherin bound beta-catenin and plakoglobin are required for cell adhesion, membrane uncomplexed beta-catenin and plakoglobin have a function in transducing the Wnt signal from the cell surface to the nucleus XREF_BIBR, XREF_BIBR, XREF_BIBR.

In addition, KLK8-induced p53 activation promoted the pro-fibrotic reprogramming by TGF-β1 via the cooperation of p53 with Smads in endothelial cells.

As shown in Figure 11A , it was found that high glucose led to a significant increase in the association of plakoglobin with p53 , which was blocked by KLK8 knockdown .

In addition, KLK8 induced p53 activation promoted the pro fibrotic reprogramming by TGF-beta1 via the cooperation of p53 with Smads in endothelial cells.

As shown in Figure XREF_FIG A, it was found that high glucose led to a significant increase in the association of plakoglobin with p53, which was blocked by KLK8 knockdown.

Taken together, these data highlight the role of KLK8 induced cooperation of p53 with Smad3 in promoting pro EndMT reprogramming by TGF-beta1 in endothelial cells.

The present study found that KLK8 overexpression significantly increased the association of p53 with Smad3, which was blocked by plakoglobin knockdown.

KLK8 overexpression induced the association of p53 with HIF-1alpha, which was blocked by plakoglobin knockdown.

Of note, since KLK8 promotes proliferation and migration in cardiac fibroblasts, a potential contribution of KLK8 induced fibroblast activation to the development of cardiac fibrosis in the context of diabetes can not be excluded.

Cell proliferation assay and Ki67 immunostaining revealed that KLK8 overexpression markedly enhanced the proliferation of cardiac fibroblasts.

These findings indicate that Sp-1 mediates high glucose induced upregulation of KLK8 in endothelial cells.

Sp-1 mediates high glucose induced upregulation of KLK8 in endothelial cells.

We then generated a KLK8 report gene by cloning the promoter of human KLK8 into the pGL3 vector , thereby further confirming that high glucose stimulates KLK8 expression through the Sp-1 site in the KLK8 gene .

It was found that the mRNA and protein expression levels of KLK8 were significantly upregulated in a dose-dependent manner in high glucose-treated HCAECs compared with those of normal glucose-treated HCAECs ( Figure 8A-B ) , suggesting that high glucose stimulated KLK8 expression at the transcriptional level .

A major implication of high glucose induced KLK8 upregulation is that KLK8 targeted therapy would have clinical relevance for patients with diabetic myopathy.

These findings indicate that Sp-1 mediates high glucose induced upregulation of KLK8 in endothelial cells.

Sp-1 mediates high glucose induced upregulation of KLK8 in endothelial cells.

KLK8 siRNA not only led to a significant decrease of KLK8 expression in HCAECs, but also blocked the high glucose induced upregulation of KLK8.

High glucose may promote the plakoglobin dependent cooperation of p53 with HIF-1alpha and Smad3, subsequently increasing the expression of TGF-beta1 and the pro EndMT target genes of the TGF-beta1 and Smad signaling pathway in a KLK8 dependent manner both in vitro and in vivo.

Taken together, these in vitro and in vivo results suggest that high glucose may promote plakoglobin dependent cooperation of p53 with HIF-1alpha and Smad3, subsequently increasing the expression of TGF-beta1 and its pro EndMT target genes in a KLK8 dependent manner.

High glucose promotes plakoglobin dependent cooperation of p53 with HIF-1alpha and Smad, subsequently increasing the expression of TGF-beta1 and its pro fibrotic target genes in a KLK8 dependent manner.

The in vitro and in vivo findings further demonstrated that high glucose may promote plakoglobin dependent cooperation of p53 with HIF-1alpha and Smad3, subsequently increasing the expression of TGF-beta1 and the pro EndMT target genes of the TGF-beta1 and Smad signaling pathway in a KLK8 dependent manner.

The in vitro and in vivo findings further demonstrated that high glucose may promote plakoglobin dependent cooperation of p53 with HIF-1alpha and Smad3, subsequently increasing the expression of TGF-beta1 and the pro EndMT target genes of the TGF-beta1 and Smad signaling pathway in a KLK8 dependent manner.

Downregulation of PTEN expression or inhibiting its biologic activity improves heart function, promotes cardiomyocytes proliferation, reduces cardiac fibrosis as well as dilation, and inhibits apoptosis following ischemic stress such as myocardial infarction.

Downregulation of PTEN expression or inhibiting its biologic activity improves heart function , promotes cardiomyocytes proliferation , reduces cardiac fibrosis as well as dilation , and inhibits apoptosis following ischemic stress such as myocardial infarction .

Collectively, these data indicate that both CYP2C19 and CYP2C9 are important contributors in CBD metabolism to the active metabolite 7-OH-CBD.

Collectively, these data indicate that both CYP2C19 and CYP2C9 are important contributors in CBD metabolism to the active metabolite 7-OH-CBD.

Collectively, these data indicate that both CYP2C19 and CYP2C9 are important contributors in CBD metabolism to the active metabolite 7-OH-CBD.

Collectively, these data indicate that both CYP2C19 and CYP2C9 are important contributors in CBD metabolism to the active metabolite 7-OH-CBD.

Moreover, GAS5 and Smad4 overexpression inhibited LPS- induced chondrocytes apoptosis, while miR-146a overexpression played an opposite role and attenuated the effects of GAS5 and Smad4 overexpression on cell apoptosis.

PTEN and PTPs all antagonize the insulin signaling as they directly interact with PI3K and IR [XREF_BIBR], and both consist of a cysteine residue in the active site that is highly susceptible to H 2 O 2 -induced oxidation.

The C2 domain ( amino acids 186-351 ) can bind phospholipid membrane independent of calcium because it lacks the canonical Ca2 + chelating residues in vitro , which makes PTEN inhibit cell migration [ 31 ] .

For instance, H 2 O 2 can induce PTEN oxidation, which inactivates PTEN phosphatase function by establishing a Cys 124 -Cys 71 disulfide bond [XREF_BIBR].

For example, the Parkinson disease protein 7 (PARK7) was found to repress the PTEN phosphatase function by binding to PTEN.

Recently, it has been found that the impairment of PARK2 can induce the suppression of PTEN by S nitrosylation through increase the level of NO [XREF_BIBR].

Recently , it has been found that the impairment of PARK2 can induce the suppression of PTEN by S-nitrosylation through increase the level of NO [ 55 ] .

Besides, Prx II deficient MEFs induced PTEN oxidation and increased PI3K and Akt activation when exposed to insulin, which leads to an increase the insulin sensitivity.

Prx III-deficiency also induces the augmentation in both PTEN oxidation and Trx dimerization.

In addition, p300-CREB-binding protein (CBP) has been shown to acetylate PTEN at Lys 402 in the PDZ-binding motif.

It has been demonstrated that Prx I can preserve and promote the tumor-suppressive function of PTEN by preventing oxidation of PTEN under benign oxidative stress via direct interaction.

Also, Prx II deficient cells increased PTEN oxidation and insulin sensitivity.

To examine whether FoxO3a is required for PTEN mediated negative regulation of autophagy, we analyzed the mRNA levels of ATG5, ATG7, and ATG12 in the ipsilateral hippocampus post-ICH.

In the present study, PTEN inhibition not only reduced the levels of autophagy related proteins but also activated the PI3K and AKT pathway in the ipsilateral hippocampus after ICH.

Functionally, our findings confirmed that inhibition of PTEN by PTEN siRNA or specific inhibitor not only ameliorated secondary hippocampal injury but also promoted hippocampal-dependent cognition and memory recovery, suggesting important neuroprotective effects against hemorrhagic insults.

Specifically, PTEN antagonized the PI3K and AKT signaling and downstream effector FoxO3a phosphorylation and subsequently enhanced nuclear translocation of FoxO3a to drive proautophagy gene program, but these changes were diminished upon PTEN inhibition.

Mechanistically, blockage of PTEN could enhance FoxO3a phosphorylation modification to restrict its nuclear translocation and ATG transcription via activating the PI3K and AKT pathway, leading to the suppression of the autophagic program.

Inhibition of PTEN Ameliorates Secondary Hippocampal Injury and Cognitive Deficits after Intracerebral Hemorrhage : Involvement of AKT / FoxO3a / ATG-Mediated Autophagy .

Inhibition of PTEN Ameliorates Secondary Hippocampal Injury and Cognitive Deficits after Intracerebral Hemorrhage : Involvement of AKT / FoxO3a / ATG-Mediated Autophagy Spontaneous intracerebral hemorrhage ( ICH ) commonly causes secondary hippocampal damage and delayed cognitive impairments , but the mechanisms remain elusive .

According to these data, we speculate that posthemorrhagic PTEN elevation triggers the nuclear accumulation of FoxO3a and subsequent transcriptional activation of ATGs, resulting in sequential activation of autophagy.