ALK5 inhibition blocks TGFβ-induced CCN1 expression in human foreskin fibroblasts

The potent profibrotic cytokine TGFβ induces connective tissue growth factor (CCN2/CTGF) is induced in fibroblasts in a fashion sensitive to SB-431542, a specific pharmacological inhibitor of TGFβ type I receptor (ALK5). In several cell types, TGFβ induces CCN1 but suppresses CCN3, which opposes CCN1/CCN2 activities. However, whether SB-431542 alters TGFβ-induced CCN1 or CCN3 in human foreskin fibroblasts in unclear. Here we show that TGFβ induces CCN1 but suppresses CCN3 expression in human foreskin fibroblasts in a SB-431542-sensitive fashion. These results emphasize that CCN1/CCN2 and CCN3 are reciprocally regulated and support the notion that blocking ALK5 or addition of CCN3 may be useful anti-fibrotic approaches.     

Repeated exposure of human fibroblasts to ionizing radiation reveals an adaptive response that is not mediated by interleukin-6 or TGF-β

Exposing cells to a low dose can protect them against a subsequent higher exposure. This phenomenon is known as adaptive response and is frequently observed in a variety of cells. Even though similarities are suspected with other non-targeted effects, such as bystander effects, the exact mechanism behind adaptive response is not fully clarified. In this study human primary fibroblasts were tested for their response to ionizing radiation (IR) after administrating a low priming dose (0.1-0.5Gy). Both the abundance of γH2AX as a marker for double-stranded breaks and the levels of cytokines, secreted in the medium, were monitored in time. Upon challenge, IR-primed cells showed modified γH2AX spot size distributions and altered repair kinetics, consistent with an adaptive response. In addition, 24h after priming with IR, four cytokines were significantly upregulated in the medium - GM-CSF (1.33×); IL6 (4.24×); IL8 (1.33×); TGF-β (1.46×). In order to mimick the protective effect of IR priming, we primed the cells with either IL6 or TGF-β. This did not elicit an altered γH2AX response as observed in IR-primed cells, indicating that the adaptive response in these primary fibroblasts is regulated in an IL-6 and TGF-β independent manner.     

Simvastatin inhibits TGFβ1-induced fibronectin in human airway fibroblasts

Background

Bronchial fibroblasts contribute to airway remodelling, including airway wall fibrosis. Transforming growth factor (TGF)-β1 plays a major role in this process. We previously revealed the importance of the mevalonate cascade in the fibrotic response of human airway smooth muscle cells. We now investigate mevalonate cascade-associated signaling in TGFβ1-induced fibronectin expression by bronchial fibroblasts from non-asthmatic and asthmatic subjects.

Methods

We used simvastatin (1-15 μM) to inhibit 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate. Selective inhibitors of geranylgeranyl transferase-1 (GGT1; GGTI-286, 10 μM) and farnesyl transferase (FT; FTI-277, 10 μM) were used to determine whether GGT1 and FT contribute to TGFβ1-induced fibronectin expression. In addition, we studied the effects of co-incubation with simvastatin and mevalonate (1 mM), geranylgeranylpyrophosphate (30 μM) or farnesylpyrophosphate (30 μM).

Results

Immunoblotting revealed concentration-dependent simvastatin inhibition of TGFβ1 (2.5 ng/ml, 48 h)-induced fibronectin. This was prevented by exogenous mevalonate, or isoprenoids (geranylgeranylpyrophosphate or farnesylpyrophosphate). The effects of simvastatin were mimicked by GGTI-286, but not FTI-277, suggesting fundamental involvement of GGT1 in TGFβ1-induced signaling. Asthmatic fibroblasts exhibited greater TGFβ1-induced fibronectin expression compared to non-asthmatic cells; this enhanced response was effectively reduced by simvastatin.

Conclusions

We conclude that TGFβ1-induced fibronectin expression in airway fibroblasts relies on activity of GGT1 and availability of isoprenoids. Our results suggest that targeting regulators of isoprenoid-dependent signaling holds promise for treating airway wall fibrosis.     

Differential expression, function and response to inflammatory stimuli of 11β-hydroxysteroid dehydrogenase type 1 in human fibroblasts: a mechanism for tissue-specific regulation of inflammation

Stromal cells such as fibroblasts play an important role in defining tissue-specific responses during the resolution of inflammation. We hypothesized that this involves tissue-specific regulation of glucocorticoids, mediated via differential regulation of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Expression, activity and function of 11β-HSD1 was assessed in matched fibroblasts derived from various tissues (synovium, bone marrow and skin) obtained from patients with rheumatoid arthritis or osteoarthritis. 11β-HSD1 was expressed in fibroblasts from all tissues but mRNA levels and enzyme activity were higher in synovial fibroblasts (2-fold and 13-fold higher mRNA levels in dermal and synovial fibroblasts, respectively, relative to bone marrow). Expression and activity of the enzyme increased in all fibroblasts following treatment with tumour necrosis factor-α or IL-1β (bone marrow: 8-fold and 37-fold, respectively, compared to vehicle; dermal fibroblasts: 4-fold and 14-fold; synovial fibroblasts: 7-fold and 31-fold; all P < 0.01 compared with vehicle). Treatment with IL-4 or interferon-γ was without effect, and there was no difference in 11β-HSD1 expression between fibroblasts (from any site) obtained from patients with rheumatoid arthritis or osteoarthritis. In the presence of 100 nmol/l cortisone, IL-6 production – a characteristic feature of synovial derived fibroblasts – was significantly reduced in synovial but not dermal or bone marrow fibroblasts. This was prevented by co-treatment with an 11β-HSD inhibitor, emphasizing the potential for autocrine activation of glucocorticoids in synovial fibroblasts. These data indicate that differences in fibroblast-derived glucocorticoid production (via the enzyme 11β-HSD1) between cells from distinct anatomical locations may play a key role in the predeliction of certain tissues to develop persistent inflammation.     

Inhibitory role of Id1 on TGF-β-induced collagen expression in human dermal fibroblasts

Inhibitor of DNA binding 1 (Id1) is a basic helix-loop-helix (bHLH) protein that has a variety of functional roles in cellular events including differentiation, cell cycle and cancer development. In addition, it has been demonstrated that Id1 is related with TGF-β and Smad signaling in various biological conditions. In this study, we investigated the effect of Id1 on TGF-β-induced collagen expression in human dermal fibroblasts. When Id1-b isoform was overexpressed, TGF-β-induced collagen expression was markedly inhibited. Consistent with this result, Id1-b significantly inhibited TGF-β-induced collagen gel contraction. In addition, Id1-b inhibited TGF-β-induced phosphorylation of Smad2 and Smad3. Finally, immunohistochemistry showed that Id1 expression was decreased in fibrotic skin diseases while TGF-β signaling was increased. Together, these results suggest that Id1 is an inhibitory regulator on TGF-β-induced collagen expression in dermal fibroblasts.     

Hydrogen sulfide suppresses transforming growth factor-β1-induced differentiation of human cardiac fibroblasts into myofibroblasts

In heart disease, transforming growth factor-β1(TGF-β1) converts fibroblasts into myofibroblasts, which synthesize and secrete fibrillar type I and III collagens. The purpose of the present study was to investigate how hydrogen sulfide(H2S) suppresses TGF-β1-induced differentiation of human cardiac fibroblasts to myofibroblasts. Human cardiac fibroblasts were serum-starved in fibroblast medium for 16 h before exposure to TGF-β1(10 ng m L-1) for 24 h with or without sodium hydrosulfide(Na HS, 100 μmol L-1, 30 min pretreatment) treatment. Na HS, an exogenous H2 S donor, potently inhibited the proliferation and migration of TGF-β1-induced human cardiac fibroblasts and regulated their cell cycle progression. Furthermore, Na HS treatment led to suppression of fibroblast differentiation into myofibroblasts, and reduced the levels of collagen, TGF-β1, and activated Smad3 in TGF-β1-induced human cardiac fibroblasts in vitro. We therefore conclude that H2 S suppresses TGF-β1-stimulated conversion of fibroblasts to myofibroblasts by inhibiting the TGF-β1/Smad3 signaling pathway, as well as by inhibiting the proliferation, migration, and cell cycle progression of human cardiac myofibroblasts. These effects of H2 S may play significant roles in cardiac remodeling associated with heart failure.     

Cellular responses to TGFβ and TGFβ receptor expression in human colonic epithelial cells require CaSR expression and function

CaSR and TGFβ are robust promoters of differentiation in the colonic epithelium. Loss of cellular responses to TGFβ or loss of CaSR expression is tightly linked to malignant progression. Human colonic epithelial CBS cells, originally developed from a differentiated human colon tumor, retain CaSR expression and function, TGFβ responsiveness and TGFβ receptor expression. Thus, these cells offer a unique opportunity in determining the functional linkage (if any) between CaSR and TGFβ. Knocking down CaSR expression abrogated TGFβ-mediated cellular responses and attenuated the expression of TGFβ receptors. Ca²⁺ or vitamin D treatment induced CaSR expression with a concurrent up-regulation of TGFβ receptor expression. Ca²⁺ or vitamin D, however, did not induce CaSR in CaSR knocked down cells and without CaSR; there was no up-regulation of TGFβ receptor. It is concluded that TGFβ receptor expression and TGFβ mediated responses requires CaSR expression and function.     

A conserved TGFβ1/HuR feedback circuit regulates the fibrogenic response in fibroblasts

Persistent fibroblast activation in wound repair is believed to be the key reason for fibrosis and transforming growth factor (TGF)β is considered as one of the key mediators for the fibrogenic response, with the detailed mechanism largely unknown. Here we found that TGFβ1 treatment could induce a significant increase of endogenous TGFβ1 expression by enhancing the mRNA stability in cardiac fibroblasts. Further study revealed that TGFβ1 treatment translocated the nuclear HuR into cytoplasm, which in turn bound the ARE in the 3'UTR of TGFβ1 and increased the mRNA stability as seen from the RNA-IP and reporter assay. Knockdown of HuR decreased the endogenous expression of TGFβ1 under exogenous TGFβ1 treatment, simultaneously with the decrease of Col1a, Col3a and fibronectin expression. Our study here established a TGFβ1/HuR feedback circuit regulating the fibrogenic response in fibroblasts, and targeting this feedback loop is of great potential to control fibrosis.     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

Surface expression of Hsp70B’ in response to proteasome inhibition in human colon cells

Hsp70B’ was expressed on the surface of HT-29 and CRL-1809 but not SW-480 human colon cell lines in response to proteasome inhibition as detected using flow cytometry. Surface expression was not detected under non-stress conditions nor was heat shock an inducer of surface expression in the three cell lines tested. Phylogenetic analysis indicated that the Hsp70B’ protein sequence was most closely related to another major inducible human Hsp70, Hsp72. Hsp70B’ appeared to be recently diverged, as homologs for Hsp70B’ have not been found in rodents. Hsp72 and Hsp70B’ shared 100% amino acid sequence identity in their predicted peptide-binding regions suggesting that they bind the same peptide substrates, perhaps in extracellular antigen presentation. Amino acid sequence differences were concentrated in the lid regions and the C-terminal domains raising the possibility that Hsp72 and Hsp70B’ bind different co-chaperones or cell surface receptors.     

Increased expression of TGF-β1 reduces tumor growth of human U-87 Glioblastoma Cells in vivo

The role that transforming growth factor β1 (TGF-β1) plays in influencing growth of glioma cells is somewhat controversial. To further understand the potential growth-regulatory effects of TGF-β1,we constructed an animal astroglial tumor model by injecting either wild-type or virally transduced human U-87 glioblastoma cells into nude rat brains. Wild type U-87 cells produced very low amounts of TGF-β1 and were highly tumorigenic. In contrast, U-87 cells transduced to express high levels of TGF-β1 showed reduced tumor size in vivo, in a dose-dependent manner. This reduction in tumor size was not due to either decreased vascularity or increased apoptosis. To test whether TGF-β1 overproduction inhibited tumor growth through an autocrine mechanism, the highest TGF-β1 producing cells were then double transduced with a vector expressing the kinase-truncated type II TGF-β receptor. Cells expressing high levels of truncated TGF-β receptor were less sensitive to TGF-β1 mediated growth inhibition in vitro and produced more aggressive tumors in vivo. The data suggest that the degree of tumorigenicity of the U-87 high-grade glioblastoma cell line may be associated with correspondingly low level of production of TGF-β1. These results also would tend to support the possibility that TGF-β1 may be useful in treating some high-grade gliomas.     

Down-regulation of Id-1 expression is associated with TGFβ1-induced growth arrest in prostate epithelial cells

Transforming growth factor β1 (TGFβ1) plays important roles in the regulation of cell growth and differentiation in both normal and malignant prostate epithelial cells. Although certain pathways have been suggested, the mechanisms responsible for the action of TGFβ1 are not well understood. In the present study, using a human papilloma virus 16 E6/E7 immortalized prostate epithelial cell line, HPr-1, we report that TGFβ1 was able to suppress the expression of Id-1, a helix–loop–helix (HLH) protein, which plays important roles in the inhibition of cell differentiation and growth arrest. In addition, a decrease at both Id-1 mRNA and protein expression levels was associated with TGFβ1-induced growth arrest and differentiation, indicating that Id-1 may be involved in TGFβ1 signaling pathway. The fact that up-regulation of p21^WAF1, one of the downstream effectors of Id-1, was observed after exposure to TGFβ1 further indicates the involvement of Id-1 in the TGFβ1-induced growth arrest in HPr-1 cells. However, increased expression of p27^KIP1 was also observed in the TGFβ1-treated cells, suggesting that in addition to down-regulation of Id-1, other factors may be involved in the TGFβ1-induced cell growth arrest and differentiation in prostate epithelial cells. Our results provide evidence for the first time that TGFβ1 may be one of the upstream regulators of Id-1.     

Controversy surrounding the increased expression of TGFβ1 in asthma

Asthma is a waxing and waning disease that leads to structural changes in the airways, such as subepithelial fibrosis, increased mass of airway smooth muscle and epithelial metaplasia. Such a remodeling of the airways futher amplifies asthma symptoms, but its etiology is unknown. Transforming growth factor β1 is a pleiotropic cytokine involved in many fibrotic, oncologic and immunologic diseases and is believed to play an essential role in airway remodeling that occurs in asthmatic patients. Since it is secreted in an inactive form, the overall activity of this cytokine is not exclusively determined by its level of expression, but also by extensive and complex post-translational mechanisms, which are all importanin modulating the magnitude of the TGFβ1 response. Even if TGFβ1 upregulation in asthma is considered as a dogma by certain investigators in the field, the overall picture of the published litterature is not that clear and the cellular origin of this cytokine in the airways of asthmatics is still a contemporaneous debate. On the other hand, it is becoming clear that TGFβ1 signaling is increased in the lungs of asthmatics, which testifies the increased activity of this cytokine in asthma pathogenesis. The current work is an impartial and exhaustive compilation of the reported papers regarding the expression of TGFβ1 in human asthmatics. For the sake of comparison, several studies performed in animal models of the disease are also included. Inconsistencies observed in human studies are discussed and conclusions as well as trends from the current state of the litterature on the matter are proposed. Finally, the different points of regulation that can affect the amplitude of the TGFβ1 response are briefly revised and the possibility that TGFβ1 is disregulated at another level in asthma, rather than simply in its expression, is highlighted.     

Stimulation by transforming growth factor-β of epidermal growth factor-dependent growth of aged human fibroblasts: Recovery of high affinity EGF receptors and growth stimulation by EGF

Summary The stimulatory effects of transforming growth factor β (TGF-β) on epidermal growth factor (EGF)-dependent growth of adult and newborn human fibroblasts were investigated. EGF-stimulated growth in low serum of dermal fibroblasts from a 41 year-old adult (HSF-41) was less than half that of newborn foreskin fibroblasts (HFF). The EGF-stimulated growth of HFF after 55 population doublings (HFF-55) was similarly reduced. The decreased growth response to EGF of fibroblasts, agedin vivo andin vitro appeared to result principally from a decreased sensitivity to EGF due to a decreased number and affinity of high affinity EGF receptors (H-EGFR). Pre-incubation of HSF-41 and HFF-55 with 25 pM TGF-β enhanced the growth responses of these cells to EGF and increased the levels of high affinity EGF-binding by these cells Thus, the stimulation by TGF-β of EGF-dependent growth of human fibroblasts agedin vivo orin vitro is mediated by increased levels of high affinity EGF binding. This research was supported in part by a grant-in-aid for scientific research (61480388) and a special project research grant to Okayama University from the Japanese Ministry of Education, Science and Culture. Editor's statement TGF beta interaction with its receptor is known to affect EGF receptors. In this paper a functional biological association is established.