TGF‐β Stimulates Expression of Chondroitin Polymerizing Factor in Nucleus Pulposus Cells Through the Smad3, RhoA/ROCK1, and MAPK Signaling Pathways

The enzyme chondroitin polymerizing factor (ChPF) is primarily involved in extension of the chondroitin sulfate backbone required for the synthesis of sulfated glycosaminoglycan (sGAG). Transforming growth factor beta (TGF‐β) upregulates sGAG synthesis in nucleus pulposus cells; however, the mechanisms mediating this induction are incompletely understood. Our study demonstrated that ChPF expression was negatively correlated with the grade of degenerative intervertebral disc disease. Treatment of nucleus pulposus cells with TGF‐β induced ChPF expression and enhanced Smad2/3, RhoA/ROCK activation, and the JNK, p38, and ERK1/2 MAPK signaling pathways. Selective inhibitors of Smad2/3, RhoA or ROCK1/2, and knockdown of Smad3 and ROCK1 attenuated ChPF expression and sGAG synthesis induced by TGF‐β. In addition, we showed that RhoA/ROCK1 signaling upregulated ChPF via activation of the JNK pathway but not the p38 and ERK1/2 signaling pathways. Moreover, inhibitors of JNK, p38 and ERK1/2 activity also blocked ChPF expression and sGAG synthesis induced by TGF‐β in a Smad3‐independent manner. Collectively, our data suggest that TGF‐β stimulated the expression of ChPF and sGAG synthesis in nucleus pulposus cells through Smad3, RhoA/ROCK1 and the three MAPK signaling pathways. J. Cell. Biochem. 119: 566–579, 2018. © 2017 Wiley Periodicals, Inc.     

RIPK4 suppresses the TGF‐β1 signaling pathway in HaCaT cells

Receptor‐interacting serine/threonine kinase 4 (RIPK4) and transforming growth factor‐β 1 (TGF‐β1) play critical roles in the development and maintenance of the epidermis. A negative correlation between the expression patterns of RIPK4 and TGF‐β signaling during epidermal homeostasis‐related events and suppression of RIPK4 expression by TGF‐β1 in keratinocyte cell lines suggest the presence of a negative regulatory loop between the two factors. So far, RIPK4 has been shown to regulate nuclear factor‐κB (NF‐κB), protein kinase C (PKC), wingless‐type MMTV integration site family (Wnt), and (mitogen‐activated protein kinase) MAPK signaling pathways. In this study, we examined the effect of RIPK4 on the canonical Smad‐mediated TGF‐β1 signaling pathway by using the immortalized human keratinocyte HaCaT cell line. According to our results, RIPK4 inhibits intracellular Smad‐mediated TGF‐β1 signaling events through suppression of TGF‐β1‐induced Smad2/3 phosphorylation, which is reflected in the upcoming intracellular events including Smad2/3‐Smad4 interaction, nuclear localization, and TGF‐β1‐induced gene expression. Moreover, the kinase activity of RIPK4 is required for this process. The in vitro wound‐scratch assay demonstrated that RIPK4 suppressed TGF‐β1‐mediated wound healing through blocking TGF‐β1‐induced cell migration. In conclusion, our results showed the antagonistic effect of RIPK4 on TGF‐β1 signaling in keratinocytes for the first time and have the potential to contribute to the understanding and treatment of skin diseases associated with aberrant TGF‐β1 signaling.     

Crosstalk between Smad2/3 and specific isoforms of ERK in TGF‐β1‐induced TIMP‐3 expression in rat chondrocytes

This study investigated the roles of ERK1 and ERK2 in transforming growth factor‐β1 (TGF‐β1)‐induced tissue inhibitor of metalloproteinases‐3 (TIMP‐3) expression in rat chondrocytes, and the specific roles of ERK1 and ERK2 in crosstalk with Smad2/3 were investigated to demonstrate the molecular mechanism of ERK1/2 regulation of TGF‐β1 signalling. To examine the interaction of specific isoforms of ERK and the Smad2/3 signalling pathway, chondrocytes were infected with LV expressing either ERK1 or ERK2 siRNA and stimulated with or without TGF‐β1. At indicated time‐points, TIMP‐3 expression was determined by real‐time PCR and Western blotting; p‐Smad3, nuclear p‐Smad3, Smad2/3, p‐ERK1/2 and ERK1/2 levels were assessed. And then, aggrecan, type II collagen and the intensity of matrix were examined. TGF‐β1‐induced TIMP‐3 expression was significantly inhibited by ERK1 knock‐down, and the decrease in TIMP‐3 expression was accompanied by a reduction of p‐Smad3 in ERK1 knock‐down cells. Knock‐down of ERK2 had no effect on neither TGF‐β1‐induced TIMP‐3 expression nor the quantity of p‐Smad3. Moreover, aggrecan, type II collagen expression and the intensity of matrix were significantly suppressed by ERK1 knock‐down instead of ERK2 knock‐down. Taken together, ERK1 and ERK2 have different roles in TGF‐β1‐induced TIMP‐3 expression in rat chondrocytes. ERK1 instead of ERK2 can regulate TGF‐β/Smad signalling, which may be the mechanism through which ERK1 regulates TGF‐β1‐induced TIMP‐3 expression.     

Transforming Growth Factor‐β1 Modulates the Expression of Syndecan‐4 in Cultured Vascular Endothelial Cells in a Biphasic Manner

Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. Previously, we reported that transforming growth factor‐β₁ (TGF‐β₁) regulates the synthesis of a large heparan sulfate proteoglycan, perlecan, and a small leucine‐rich dermatan sulfate proteoglycan, biglycan, in vascular endothelial cells depending on cell density. Recently, we found that TGF‐β₁ first upregulates and then downregulates the expression of syndecan‐4, a transmembrane heparan sulfate proteoglycan, via the TGF‐β receptor ALK5 in the cells. In order to identify the intracellular signal transduction pathway that mediates this modulation, bovine aortic endothelial cells were cultured and treated with TGF‐β₁. Involvement of the downstream signaling pathways of ALK5—the Smad and MAPK pathways—in syndecan‐4 expression was examined using specific siRNAs and inhibitors. The data indicate that the Smad3–p38 MAPK pathway mediates the early upregulation of syndecan‐4 by TGF‐β₁, whereas the late downregulation is mediated by the Smad2/3 pathway. Multiple modulations of proteoglycan synthesis may be involved in the regulation of vascular endothelial cell functions by TGF‐β₁. J. Cell. Biochem. 118: 2009–2017,2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Distribution of TGF‐β isoforms and signaling intermediates in corneal fibrotic wound repair

In this study, temporal and spatial distribution of three TGF‐β isoforms and their downstream signaling pathways including pSmad2 and p38MAPK were examined during fibrotic wound repair. In normal chick corneas, TGF‐β1, ‐2, and ‐3 were weakly detected in Bowman's layer (BL). In healing corneas, TGF‐β1 was primarily deposited in the fibrin clot and the unwounded BL. TGF‐β2 was highly expressed in healing epithelial and endothelial cells, and numerous active fibroblasts/myofibroblasts. TGF‐β3 was mainly detected in the unwound region of basal epithelial cells. α‐Smooth muscle actin (α‐SMA) was initially appeared in the posterior region of repairing stroma at day 3, and was detected in the entire healing stroma by day 7. Notably, α‐SMA was absent in the central region of healing stroma by day 14, and its staining pattern was similar to those of TGF‐β2 and p38MAPK. By contrast, pSmad2 was mainly detected in the fibroblasts. In normal cornea, laminin was mainly detected in both epithelial basement membrane (BM) and Descemet's membrane (DM). By contrast to reconstitution of the BM in the wound region, the DM was not repaired although endothelial layer was regenerated, indicating that high levels of TGF‐β2 were released into the posterior region of healing stroma on day 14. High levels of α‐SMA staining, shown in cultured repair stromal cells from healing corneas on day 14 and in TGF‐β2 treated normal stromal cells, were significantly reduced by p38MAPK inhibition. Collectively, this study suggests that TGF‐β2‐mediated myofibroblast transformation is mediated, at least partly, by the p38MAPK pathway in vivo. J. Cell. Biochem. 108: 476–488, 2009. © 2009 Wiley‐Liss, Inc.     

Comparative proteome analysis of TGF‐β1‐induced fibrosis processes in normal rat kidney interstitial fibroblast cells in response to ascofuranone

Transforming growth factor‐β1 (TGF‐β1) has a wide range of biological functions such as the regulation of cell growth, differentiation, and immunological response in various types of cells. Particularly, TGF‐β1 induces plasminogen activator inhibitor‐1 (PAI‐1) as a major target protein. PAI‐1 is associated with fibrosis, thrombosis, and metabolic disorders. In this study, to identify proteins potentially involved in TGF‐β1‐induced fibrosis processes, we performed a proteomic analysis of TGF‐β1‐induced normal rat kidney cells exposed to ascofuranone (AF). In these cells, we detected 1500 proteins, with 74 differentially expressed proteins identified by MALDI‐TOF and reference to the NCBI and Swiss‐Prot databases, including PAI‐1, peroxisome prdifesator‐activated receptor, prohibitin, glutamate formyltransferase, LIM domain protein 1, LASP‐1, porphobilinogen deaminase, and peroxiredoxin 2. We also found that AF suppresses expression of profibrotic factors induced by TGF‐β in renal fibroblasts, including matrix proteins and PAI‐1. AF was also shown to inhibit selectively phosphorylation of epidermal growth factor receptor, and downstream kinases such as extracellular signal‐regulated kinase 1/2 (ERK‐1/2). Further ongoing analysis of fibrosis‐related proteins will determine AF's potential for application in fibrotic diseases and therapeutics.     

Angiotensin II increases mRNA levels of all TGF‐β isoforms in quiescent and activated rat hepatic stellate cells

AII (angiotensin II) is a vasoactive peptide that plays an important role in the development of liver fibrosis mainly by regulating profibrotic cytokine expression such as TGF‐β (transforming growth factor‐β). Activated HSCs (hepatic stellate cells) are the major cell type responsible for ECM (extracellular matrix) deposition during liver fibrosis and are also a target for AII and TGF‐β actions. Here, we studied the effect of AII on the mRNA levels of TGF‐β isoforms in primary cultures of rat HSCs. Both quiescent and activated HSCs were stimulated with AII for different time periods, and mRNA levels of TGF‐β1, TGF‐β2 and TGF‐β3 isoforms were evaluated using RNaseI protection assay. The mRNA levels of all TGF‐β isoforms, particularly TGF‐β2 and TGF‐β3, were increased after AII treatment in activated HSCs. In addition, activated HSCs were able to produce active TGF‐β protein after AII treatment. The mRNA expression of TGF‐β isoforms induced by AII required both ERK1/2 and Nox (NADPH oxidase) activation but not PKC (protein kinase C) participation. ERK1/2 activation induced by AII occurs via AT1 receptors, but independently of either PKC and Nox activation or EGFR (epidermal growth factor receptor) transactivation. Interestingly, AII has a similar effect on TGF‐β expression in quiescent HSCs, although it has a smaller but significant effect on ERK1/2 activation in these cells.     

Comparing the binding properties of peptides mimicking the Envelope protein of SARS‐CoV and SARS‐CoV‐2 to the PDZ domain of the tight junction‐associated PALS1 protein

The Envelope protein (E) is one of the four structural proteins encoded by the genome of SARS‐CoV and SARS‐CoV‐2 Coronaviruses. It is an integral membrane protein, highly expressed in the host cell, which is known to have an important role in Coronaviruses maturation, assembly and virulence. The E protein presents a PDZ‐binding motif at its C‐terminus. One of the key interactors of the E protein in the intracellular environment is the PDZ containing protein PALS1. This interaction is known to play a key role in the SARS‐CoV pathology and suspected to affect the integrity of the lung epithelia. In this paper we measured and compared the affinity of peptides mimicking the E protein from SARS‐CoV and SARS‐CoV‐2 for the PDZ domain of PALS1, through equilibrium and kinetic binding experiments. Our results support the hypothesis that the increased virulence of SARS‐CoV‐2 compared to SARS‐CoV may rely on the increased affinity of its Envelope protein for PALS1.     

Troxerutin downregulates C/EBP‐β gene expression via modulating the IFNγ‐ERK1/2 signaling pathway to ameliorate rotenone‐induced retinal neurodegeneration

Troxerutin, a natural flavonoid guards against oxidative stress and apoptosis with a high capability of passing through the blood‐brain barrier. Our aim was to investigate the role of troxerutin in experimentally induced retinal neurodegeneration by modulating the interferon‐gamma (IFNγ)‐extracellular signal‐regulated kinases 1/2 (ERK1/2)‐CCAAT enhancer‐binding protein β (C/EBP‐β) signaling pathway. Three groups of rats (10 each group) were included. Group I (control group), group II (rotenone treated group): the rats were injected subcutaneously with a single rotenone dosage of 3 mg/kg repeated every 48 hours for 60 days to trigger retinal neurodegeneration. Group III (troxerutin‐treated group): rats received troxerutin (150 mg/kg/day) by oral gavage 1 hour before rotenone administration. A real‐time polymerase chain reaction technique was applied to measure messenger RNA (mRNA) levels of retinal C/EBP‐β. Enzyme‐linked immunosorbent assay technique was utilized to assay tumor necrosis factor‐α (TNF‐α), IFNγ, and ERK1/2 levels. Finally, reactive oxygen species (ROS), as well as carbonylated protein (CP) levels, were assessed spectrophotometrically. Improved retinal neurodegeneration by downregulation of C/EBP‐β mRNA gene expression, also caused a significant reduction of TNF‐α, IFNγ, ERK1/2 as well as ROS and CP levels compared with the diseased group. These findings could hold promise for the usage of troxerutin as a protective agent against rotenone‐induced retinal neurodegeneration.     

Effect of budesonide on TGF‐β1‐enhanced VEGF production by lung fibroblasts

VEGF (vascular endothelial growth factor) is a potent proangiogenic cytokine, and vascular change is one of the characteristic features of airway remodelling. Since the glucocorticoids have shown antifibrosis properties, we sought to investigate whether budesonide, a widely used glucocorticoid in clinical practice, could attenuate TGF‐β1 (transforming growth factor‐β1)‐induced VEGF production by HFL‐1 (human lung fibroblasts). HFL‐1 fibroblasts were treated with various concentrations of budesonide (10^?11 M, 10^?9 M and 10^?7 M) in the absence or presence of TGF‐β1. Postculture media were collected for ELISA of VEGF at the indicated times. The cell lysates were subjected to Western blotting analysis to test TGF‐β1/Smad and MAP (mitogen‐activated protein) kinase signalling activation, respectively. The results suggested that budesonide pretreatment reduced the significant increase of VEGF release induced by TGF‐β1 in HFL‐1 fibroblasts in a dose‐dependent manner, and suppressed the increase of phospho‐Smad3 and phosphor‐ERK (extracellular signal‐regulated kinase) protein levels. In conclusion, budesonide may reduce TGF‐β1‐induced VEGF production in the lung, probably through the Smad/ERK signalling pathway and, thus, may provide new sight into the molecular mechanism underlying glucocorticoid therapy for airway inflammatory diseases.     

β‐Catenin and Rho GTPases as downstream targets of TGF‐β1 during pulp repair

TGF‐β1 (transforming growth factor‐β1) plays a central role in regulating proliferation, migration and differentiation of dental pulp cells during the repair process after tooth injury. Our previous study showed that p38 mitogen‐activated protein kinase may act downstream of TGF‐β1 signalling to effect the differentiation of dental pulp cells. However, the molecular mechanisms that trigger and regulate the process remain to be elucidated. TGF‐β1 interacts with signalling pathways such as Wnt/β‐catenin and Rho to induce diverse biological effects. TGF‐β1 activates β‐catenin signalling, increases β‐catenin nuclear translocation and interacts with LEF/TCF to regulate gene expression. Morphologic changes in response to TGF‐β1 are associated with activation of Rho GTPases, but are abrogated by inhibitors of Rho‐associated kinase, a major downstream target of Rho. These results suggest that the Wnt/β‐catenin and Rho pathways may mediate the downstream events of TGF‐β1 signalling.     

Systematic profiling of ACE2 expression in diverse physiological and pathological conditions for COVID‐19/SARS‐CoV‐2

Recent retrospective studies of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) disease (COVID‐19) revealed that the patients with common comorbidities of cancers and chronic diseases face significantly poorer clinical outcomes than those without. Since the expression profile of ACE2, a crucial cell entry receptor for SARS‐CoV‐2, could indicate the susceptibility to SARS‐CoV‐2 infection, here we systematically dissected ACE2 expression using large‐scale multi‐omics data from 30 organs/tissues, 33 cancer types and some common chronic diseases involving >28 000 samples. It was found that sex and age could be correlated with the susceptibility of SARS‐CoV‐2 infection for certain tissues. Strikingly, ACE2 was up‐regulated in cervical squamous cell carcinoma and endocervical adenocarcinoma, colon adenocarcinoma, oesophageal carcinoma, kidney renal papillary cell carcinoma, lung adenocarcinoma and uterine corpus endometrial carcinoma compared to controls. Furthermore, the patients with common chronic diseases regarding angiocardiopathy, type 2 diabetes, liver, pneumonia and hypertension were also with higher ACE2 expression compared to related controls, which were validated using independent data sets. Collectively, our study may reveal a novel important mechanism that the patients with certain cancers and chronic diseases may express higher ACE2 expression compared to the individuals without diseases, which could lead to their higher susceptibility to multi‐organ injury of SARS‐CoV‐2 infection.