CD147 mediates the CD44s-dependent differentiation of myofibroblasts driven by transforming growth factor-β1

Progressive fibrosis leads to loss of organ function and affects many organs as a result of excessive extracellular matrix production. The ubiquitous matrix polysaccharide hyaluronan (HA) is central to this through association with its primary receptor, CD44, which exists as standard CD44 (CD44s) or multiple splice variants. Mediators such as profibrotic transforming growth factor (TGF)-β1 and proinflammatory interleukin (IL)-1β are widely associated with fibrotic progression. TGF-β1 induces myofibroblast differentiation, while IL-1β induces a proinflammatory fibroblast phenotype that promotes fibroblast binding to monocyte/macrophages. CD44 expression is essential for both responses. Potential CD44 splice variants involved, however, are unidentified. The TGF-β1-activated CD44/epidermal growth factor receptor complex induces differentiation of metastatic cells through interactions with the matrix metalloproteinase inducer, CD147. This study aimed to determine the CD44 variants involved in TGF-β1- and IL-1β-mediated responses and to investigate the potential profibrotic role of CD147. Using immunocytochemistry and quantitative PCR, standard CD44s were shown to be essential for both TGF-β1-induced fibroblast/myofibroblast differentiation and IL-1β-induced monocyte binding. Co-immunoprecipitation identified that CD147 associated with CD44s. Using CD147-siRNA and confocal microscopy, we also determined that incorporation of the myofibroblast marker, αSMA, into F-actin stress fibers was prevented in the absence of CD147 and myofibroblast-dependent collagen gel contraction was inhibited. CD147 did not associate with HA, but removal of HA prevented the association of CD44s with CD147 at points of cell–cell contact. Taken together, our data suggest that CD44s/CD147 colocalization is essential in regulating the mechanical tension required for the αSMA incorporation into F-actin stress fibers that regulates myofibroblast phenotype.     

Active transforming growth factor-β in human melanoma cell lines: No evidence for plasmin-related activation of latent TGF-β

Cultured human melanoma cells were found to secrete TGF-β mostly in latent biologically inactive form but in addition five of six melanoma cell lines studied produced in conditioned culture medium active TGF-β in the range from 370 to 610 pg per 10⁶ cells per 24 h. A distinct characteristic of these melanoma cell lines is that they form active surface-bound plasmin by the activation of plasminogen with surface-bound tissue-type plasminogen activator. The present study was performed to assess the role of plasmin in the process of latent TGF-β activation in the melanoma cell lines. No direct correlation was found between cell-associated plasmin activity and the amount of active TGF-β present in the conditioned medium of individual cell lines. The melanoma cell lines exhibited diverse responses to exogenous active TGF-β1; three cell lines were growth-stimulated, two were growth-inhibited, and one had a very low sensitivity to the growth factor. The active TGF-β produced by the melanoma cells was found to inhibit the natural killer cell function of peripheral blood lymphocytes, suggesting that it may have an immunosuppressive effect and a role in the development of melanomas. © 1996 Wiley-Liss, Inc.     

The miR-302c/transforming growth factor-β receptor type-2 axis modulates interleukin-1β-induced degenerative changes in osteoarthritic chondrocytes

Chondrocyte production of catabolic and inflammatory mediators participating in extracellular matrix degradation has been regarded as a central event in osteoarthritis (OA) development. During OA pathogenesis, interleukin-1β (IL-1β) decreases the mRNA expression and protein levels of transforming growth factor-β receptor type-2 (TGFBR2), thus disrupting transforming growth factor-β signaling and promoting OA development. In the present study, we attempted to identify the differentially expressed genes in OA chondrocytes upon IL-1β treatment, investigate their specific roles in OA development, and reveal the underlying mechanism. As shown by online data analysis and experimental results, TGFBR2 expression was significantly downregulated in IL-1β-treated human primary OA chondrocytes. IL-1β treatment induced degenerative changes in OA chondrocytes, as manifested by increased matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 proteins, decreased Aggrecan and Collagen II proteins, and suppressed OA chondrocyte proliferation. These degenerative changes were significantly reversed by TGFBR2 overexpression. miR-302c expression was markedly induced by IL-1β treatment in OA chondrocytes. miR-302c suppressed the expression of TGFBR2 via direct binding to its 3′- untranslated region. Similar to TGFBR2 overexpression, miR-302c inhibition significantly improved IL-1β-induced degenerative changes in OA chondrocytes. Conversely, TGFBR2 silencing enhanced IL-1β-induced degenerative changes and significantly reversed the effects of miR-302c inhibition in response to IL-1β treatment. In conclusion, the miR-302c/TGFBR2 axis could modulate IL-1β-induced degenerative changes in OA chondrocytes and might become a novel target for OA treatment.Electronic supplementary materialThe online version of this article (10.1007/s12079-020-00591-2) contains supplementary material, which is available to authorized users.     

Overexpression of heat shock protein 70 inhibits epithelial-mesenchymal transition and cell migration induced by transforming growth factor-β in A549 cells

Heat shock protein 70 (HSP70) is a key member of the HSP family that contributes to a pre-cancerous environment; however, its role in lung cancer remains poorly understood. The present study used geranylgeranylacetone (GGA) to induce HSP70 expression, and transforming growth factor-β (TGF-β) was used to construct an epithelial-mesenchymal transition (EMT) model by stimulating A549 cells in vitro. Western Blot was performed to detect protein levels of NADPH oxidase 4 (NOX4) and the EMT-associated proteins E-cadherin and vimentin both before and after HSP70 expression. Cell morphological changes were observed, and the effect of HSP70 on cell migration ability was detected via the wound healing. The results demonstrated that GGA at 50 and 200 μmol/L could significantly induce HSP70 expression in A549 cells (P < 0.05). Furthermore, HSP70 induced by 200 μmol/L GGA significantly inhibited the changes of E-cadherin, vimentin, and cell morphology induced by TGF-β (P < 0.05), while HSP70 induced by 50 μmol/L GGA did not. The results of the wound healing assay indicated that 200 μmol/L GGA significantly inhibited A549 cell migration induced by TGF-β. Taken together, the results of the present study demonstrated that overexpression of HSP70 inhibited the TGF-β induced EMT process and changed the cell morphology and migratory ability induced by TGF-β in A549 cells.     

Transforming growth factor-β1 inhibits regeneration of renal proximal tubular cells after oxidant exposure

Transforming growth factor-β (TGF-β), a regulatory cytokine expressed in the kidney, plays a role in nephrogenic repair. This study utilized a chemical model of renal proximal tubule cellular injury and regeneration to investigate the effects of TGF-β₁ on regeneration. Confluent monolayers of rabbit renal proximal tubular cells (RPTC) in primary culture exposed to the oxidant t-butylhydroperoxide (800 μM TBHP) for 1.5 hours were 24% confluent after 24 hours. Confluency increased to 50% 4 days after TBHP exposure. Recovery of monolayer confluency was associated with increased monolayer protein but not with DNA content. Daily treatment of injured monolayers with TGF-β₁ inhibited the recovery of monolayer confluency and inhibited recovery of protein content in a concentration-dependent manner (0.02–1 ng/mL). DNA content of injured monolayers was not altered by TGF-β₁. A single treatment of injured monolayers with 0.2 ng/mL (8 pM) TGF-β₁ inhibited recovery of monolayer confluency and protein content without altering monolayer DNA content. These data show that a single 24 hour exposure to a low concentration (8 pM) of TGF-β₁ inhibits regeneration of renal proximal tubule cell monolayers following oxidative injury by inhibiting, in part, cellular migration/spreading. © 1996 John Wiley & Sons, Inc.     

Transforming growth factor-α and other growth factors stimulate cell division in olfactory epithelium in vitro

The rate of cell division in olfactory epithelium (OE) is upregulated by ablation of the olfactory bulb (Carr and Farbman, 1992), or downregulated by occlusion of a naris. We used an organ culture assay of fetal rat olfactory mucosa to study regulation of the mitotic rate. Addition of any one of three members of the epidermal growth factor (EGF) family—EGF, transforming growth factor-α (TGF-α), or amphiregulin (AR)—to a serum-free culture medium resulted in a two- to threefold increase in the number of dividing OE cells. TGF-α elicited a maximal response in a dose of 100–200 pM culture medium and was 2 orders of magnitude more potent than the other EGF family members. Addition of TGF-β1, TGF-β2, insulinlike growth factor-1 or platelet-derived growth factor to the culture medium had slightly less effect than EGF or AR, in about the same molar dose range; addition of nerve growth factor had virtually no net effect on cell division. Immunohistochemistry on adult rat OE showed that basal cells, supporting cells, and acinar cells of Bowman's glands were immunoreactive with antibody to TGF-α but not with antibody to EGF. Most growth factors upregulated division of both olfactory neuron progenitors and supporting cells. The data suggest that several growth factors, most prominently TGF-α, may participate in the mitotic regulation of OE. © 1996 John Wiley & Sons, Inc.     

Macrophage migration inhibitory factor regulates joint capsule fibrosis by promoting TGF-β1 production in fibroblasts

Joint capsule fibrosis caused by excessive inflammation results in post-traumatic joint contracture (PTJC). Transforming growth factor (TGF)-β1 plays a key role in PTJC by regulating fibroblast functions, however, cytokine-induced TGF-β1 expression in specific cell types remains poorly characterized. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in inflammation- and fibrosis-associated pathophysiology. In this study, we investigated whether MIF can facilitate TGF-β1 production from fibroblasts and regulate joint capsule fibrosis following PTJC. Our data demonstrated that MIF and TGF-β1 significantly increased in fibroblasts of injured rat posterior joint capsules. Treatment the lesion sites with MIF inhibitor 4-Iodo-6-phenylpyrimidine (4-IPP) reduced TGF-β1 production and relieved joint capsule inflammation and fibrosis. In vitro, MIF facilitated TGF-β1 expression in primary joint capsule fibroblasts by activating mitogen-activated protein kinase (MAPK) (P38, ERK) signaling through coupling with membrane surface receptor CD74, which in turn affected fibroblast functions and promoted MIF production. Our results reveal a novel function of trauma-induced MIF in the occurrence and development of joint capsule fibrosis. Further investigation of the underlying mechanism may provide potential therapeutic targets for PTJC.     

Role of the transforming growth factor-β family in the expression of cranial neural crest-specific phenotypes

Cranial and trunk neural crest cells produce different derivatives in vitro. Cranial neural crest cultures produce large numbers of cells expressing fibronectin (FN) and procollagen I (PCol I) immunoreactivities, two markers expressed by mesenchymal derivatives in vivo. Trunk neural crest cultures produce relatively few FN or PCol I immunoreactive cells, but they produce greater numbers of melanocytes than do cranial cultures. Treatment of trunk neural crest cultures with transforming growth factor-β1 (TGF-β1) stimulates them to express both FN and PCol I immunoreactivities at levels comparable to those normally seen in cranial cultures and simultaneously decreases their expression of melanin. These observations raised the possibility that endogenous TGF-β is involved in specifying differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro. Consistent with this idea, we found that treatment of cranial cultures with a function-blocking TGF-β antiserum inhibits the development of FN immunoreactive cells and stimulates the development of melanocytes. Cranial and trunk neural crest cells express approximately equal levels of TGF-β mRNA. However, trunk neural crest cells are significantly less sensitive to the FN-inducing effect of TGF-β1 than are cranial neural crest cells. These results suggest that: (1) endogenous TGF-β is required for the expression of mesenchymal phenotypes by cranial neural crest cells, and (2) differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro result in part from differences in the sensitivities of these two cell populations to TGF-β. © 1995 John Wiley & Sons, Inc.     

β-cell Smad2 null mice have improved β-cell function and are protected from diet-induced hyperglycemia

Understanding signaling pathways that regulate pancreatic β-cell function to produce, store, and release insulin, as well as pathways that control β-cell proliferation, is vital to find new treatments for diabetes mellitus. Transforming growth factor-beta (TGF-β) signaling is involved in a broad range of β-cell functions. The canonical TGF-β signaling pathway functions through intracellular smads, including smad2 and smad3, to regulate cell development, proliferation, differentiation, and function in many organs. Here, we demonstrate the role of TGF-β/smad2 signaling in regulating mature β-cell proliferation and function using β-cell-specific smad2 null mutant mice. β-cell-specific smad2-deficient mice exhibited improved glucose clearance as demonstrated by glucose tolerance testing, enhanced in vivo and ex vivo glucose-stimulated insulin secretion, and increased β-cell mass and proliferation. Furthermore, when these mice were fed a high-fat diet to induce hyperglycemia, they again showed improved glucose tolerance, insulin secretion, and insulin sensitivity. In addition, ex vivo analysis of smad2-deficient islets showed that they displayed increased glucose-stimulated insulin secretion and upregulation of genes involved in insulin synthesis and insulin secretion. Thus, we conclude that smad2 could represent an attractive therapeutic target for type 2 diabetes mellitus.     

Different N-Glycosylation Sites Reduce the Activity of Recombinant DSPAα2

Bat plasminogen activators α2 (DSPAα2) has extremely high medicinal value as a powerful natural thrombolytic protein. However, wild-type DSPAα2 has two N-glycosylation sites (N185 and N398) and its non-human classes of high-mannose-type N-glycans may cause immune responses in vivo. By mutating the N-glycosylation sites, we aimed to study the effect of its N-glycan chain on plasminogen activation, fibrin sensitivity, and to observe the physicochemical properties of DSPAα2. A logical structure design was performed in this study. Four single mutants and one double mutant were constructed and expressed in Pichia pastoris. When the N398 site was eliminated, the plasminogen activator in the mutants had their activities reduced to ~40%. When the N185 site was inactivated, there was a weak decrease in the plasminogen activation of its mutant, while the fibrin sensitivity significantly decreased by ~10-fold. Neither N-glycosylation nor deglycosylation mutations changed the pH resistance or heat resistance of DSPAα2. This study confirms that N-glycosylation affects the biochemical function of DSPAα2, which provides a reference for subsequent applications of DSPAα2.     

Transforming growth factor‐β signalling in tumour resistance to the anti‐PD‐(L)1 therapy: Updated

Low frequency of durable responses in patients treated with immune checkpoint inhibitors (ICIs) demands for taking complementary strategies in order to boost immune responses against cancer. Transforming growth factor‐β (TGF‐β) is a multi‐tasking cytokine that is frequently expressed in tumours and acts as a critical promoter of tumour hallmarks. TGF‐β promotes an immunosuppressive tumour microenvironment (TME) and defines a bypass mechanism to the ICI therapy. A number of cells within the stroma of tumour are influenced from TGF‐β activity. There is also evidence of a relation between TGF‐β with programmed death‐ligand 1 (PD‐L1) expression within TME, and it influences the efficacy of anti‐programmed death‐1 receptor (PD‐1) or anti‐PD‐L1 therapy. Combination of TGF‐β inhibitors with anti‐PD(L)1 has come to the promising outcomes, and clinical trials are under way in order to use agents with bifunctional capacity and fusion proteins for bonding TGF‐β traps with anti‐PD‐L1 antibodies aiming at reinvigorating immune responses and promoting persistent responses against advanced stage cancers, especially tumours with immunologically cold ecosystem.     

Differential activation of ErbB receptors in the rat olfactory mucosa by transforming growth factor-α and epidermal growth factor in vivo

Transforming growth factor-α (TGF-α) and epidermal growth factor (EGF) are members of the EGF family of growth factors. They have a common receptor, the EGF receptor. This belongs to the tyrosine kinase group of receptors called the ErbB receptor family. Other members are ErbB-2, ErbB-3, and ErbB-4. Binding of either ligand to the receptor elicits an increase in tyrosine kinase activity, resulting in the autophosphorylation of the receptor followed by a phosphorylation cascade of other tyrosine kinase substrates including mitogen-activated protein kinase (MAPK). TGF-α and EGF have been shown to stimulate cell division in the olfactory epithelium in vitro and may regulate cell division in vivo. To investigate whether exogenous TGF-α or EGF has a functional effect on the olfactory mucosa in vivo, 12.5–50 μg of each growth factor was administered to rats via the carotid artery. After 2 min, olfactory mucosa and liver samples were collected, homogenized, and immunoprecipitated with antibodies to the ErbB receptors. The immunoprecipitates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western immunoblotting. Using phosphotyrosine antibody, the receptors were probed for phosphorylation. Activation of MAPK was also investigated using MAPK antibody. Exogenous TGF-α activated EGFR, ErbB-2 and MAPK, whereas EGF activated only the EGFR. TGF-α was a more potent activator of EGFR than EGF. Neither ligand had an effect on ErbB-3 and ErbB-4 receptors. These effects were absent in the control animals which received the same solution without the growth factor. These results are consistent with the notion that binding of TGF-α to EGFR may play a role in olfactory cell division in vivo. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 199–210, 1998     

NR4A1 counteracts JNK activation incurred by ER stress or ROS in pancreatic β‐cells for protection

Sustained hyperglycaemia and hyperlipidaemia incur endoplasmic reticulum stress (ER stress) and reactive oxygen species (ROS) overproduction in pancreatic β‐cells. ER stress or ROS causes c‐Jun N‐terminal kinase (JNK) activation, and the activated JNK triggers apoptosis in different cells. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an inducible multi‐stress response factor. The aim of this study was to explore the role of NR4A1 in counteracting JNK activation induced by ER stress or ROS and the related mechanism. qPCR, Western blotting, dual‐luciferase reporter and ChIP assays were applied to detect gene expression or regulation by NR4A1. Immunofluorescence was used to detect a specific protein expression in β‐cells. Our data showed that NR4A1 reduced the phosphorylated JNK (p‐JNK) in MIN6 cells encountering ER stress or ROS and reduced MKK4 protein in a proteasome‐dependent manner. We found that NR4A1 increased the expression of cbl‐b (an E3 ligase); knocking down cbl‐b expression increased MKK4 and p‐JNK levels under ER stress or ROS conditions. We elucidated that NR4A1 enhanced the transactivation of cbl‐b promoter by physical association. We further confirmed that cbl‐b expression in β‐cells was reduced in NR4A1‐knockout mice compared with WT mice. NR4A1 down‐regulates JNK activation by ER stress or ROS in β‐cells via enhancing cbl‐b expression.     

Casein kinase 2 promotes the TGF-β-induced activation of α-tubulin acetyltransferase 1 in fibroblasts cultured on a soft matrix

Cell signals for growth factors depend on the mechanical properties of the extracellular matrix (ECM) surrounding the cells. Microtubule acetylation is involved in the transforming growth factor (TGF)-β-induced myofibroblast differentiation in the soft ECM. However, the mechanism of activation of α-tubulin acetyltransferase 1 (α-TAT1), a major α-tubulin acetyltransferase, in the soft ECM is not well defined. Here, we found that casein kinase 2 (CK2) is required for the TGF-β-induced activation of α-TAT1 that promotes microtubule acetylation in the soft matrix. Genetic mutation and pharmacological inhibition of CK2 catalytic activity specifically reduced microtubule acetylation in the cells cultured on a soft matrix rather than those cultured on a stiff matrix. Immunoprecipitation analysis showed that CK2α, a catalytic subunit of CK2, directly bound to the C-terminal domain of α-TAT1, and this interaction was more prominent in the cells cultured on the soft matrix. Moreover, the substitution of alanine with serine, the 236th amino acid located at the C-terminus, which contains the CK2-binding site of α-TAT1, sig-nificantly abrogated the TGF-β-induced microtubule acetylation in the soft matrix, indicating that the successful binding of CK2 and the C-terminus of α-TAT1 led to the phosphorylation of serine at the 236th position of amino acids in α-TAT1 and regulation of its catalytic activity. Taken together, our findings provide novel insights into the molecular mechanisms underlying the TGF-β-induced activation of α-TAT1 in a soft matrix.     

Butyrate inhibits IL-1β-induced inflammatory gene expression by suppression of NF-κB activity in pancreatic beta cells

Cytokine-induced beta cell dysfunction is a hallmark of type 2 diabetes (T2D). Chronic exposure of beta cells to inflammatory cytokines affects gene expression and impairs insulin secretion. Thus, identification of anti-inflammatory factors that preserve beta cell function represents an opportunity to prevent or treat T2D. Butyrate is a gut microbial metabolite with anti-inflammatory properties for which we recently showed a role in preventing interleukin-1β (IL-1β)-induced beta cell dysfunction, but how prevention is accomplished is unclear. Here, we investigated the mechanisms by which butyrate exerts anti-inflammatory activity in beta cells. We exposed mouse islets and INS-1E cells to a low dose of IL-1β and/or butyrate and measured expression of inflammatory genes and nitric oxide (NO) production. Additionally, we explored the molecular mechanisms underlying butyrate activity by dissecting the activation of the nuclear factor-κB (NF-κB) pathway. We found that butyrate suppressed IL-1β-induced expression of inflammatory genes, such as Nos2, Cxcl1, and Ptgs2, and reduced NO production. Butyrate did not inhibit IκBα degradation nor NF-κB p65 nuclear translocation. Furthermore, butyrate did not affect binding of NF-κB p65 to target sequences in synthetic DNA but inhibited NF-κB p65 binding and RNA polymerase II recruitment to inflammatory gene promoters in the context of native DNA. We found this was concurrent with increased acetylation of NF-κB p65 and histone H4, suggesting butyrate affects NF-κB activity via inhibition of histone deacetylases. Together, our results show butyrate inhibits IL-1β-induced inflammatory gene expression and NO production through suppression of NF-κB activation and thereby possibly preserves beta cell function.