High doses of TGF-β potently suppress type I collagen via the transcription factor CUX1

Abstract

Transforming growth factor-β (TGF-β) is an inducer of type I collagen, and uncontrolled collagen production leads to tissue scarring and organ failure. Here we hypothesize that uncovering a molecular mechanism that enables us to switch off type I collagen may prove beneficial in treating fibrosis. For the first time, to our knowledge, we provide evidence that CUX1 acts as a negative regulator of TGF-β and potent inhibitor of type I collagen transcription. We show that CUX1, a CCAAT displacement protein, is associated with reduced expression of type I collagen both in vivo and in vitro. We show that enhancing the expression of CUX1 results in effective suppression of type I collagen. We demonstrate that the mechanism by which CUX1 suppresses type I collagen is through interfering with gene transcription. In addition, using an in vivo murine model of aristolochic acid (AA)-induced interstitial fibrosis and human AA nephropathy, we observe that CUX1 expression was significantly reduced in fibrotic tissue when compared to control samples. Moreover, silencing of CUX1 in fibroblasts from kidneys of patients with renal fibrosis resulted in increased type I collagen expression. Furthermore, the abnormal CUX1 expression was restored by addition of TGF-β via the p38 mitogen-activated protein kinase pathway. Collectively, our study demonstrates that modifications of CUX1 expression lead to aberrant expression of type I collagen, which may provide a molecular basis for fibrogenesis.

FIGURE 1:

CUX1 suppresses type I collagen in collagen-producing cells. Diagrammatic representation of DNA constructs used for enhancing CUX1 expression in vitro is shown in (A); p200 and p75 are isoforms of CUX1. qPCR analysis was carried out to measure CUX1 and COL1A2 mRNA levels in normal kidney fibroblasts (TK173). TK173 were transfected with a p200 or p75 expression vectors or an EV. The results are expressed as fold change increase when compared to nontransfected cells, which serve as baseline (B). By using Western blotting techniques, we measured the protein level of CUX1 (both isoforms), type I collagen, and β-actin (loading control) (C). To validate our results, CUX1 and COL1A2 mRNA expression was also measured in normal lung (D) and normal skin fibroblasts (E).

FIGURE 2:

CUX1 binds to and regulates COL1A2 at the level of transcription. Two putative consensus sites for CUX1 binding were identified in the COL1A2 promoter using web-based bioinformatics analysis (Genomatix, Ann Arbor, MI). The location and sequence information of DNA probes used for EMSA is shown in (A) (probes 1 and 2). The CBF point mutation is highlighted in green (A). Three stable transfectant lines, α, β, and γ, were generated overexpressing CUX1 at different concentrations. Nuclear CUX1 and lamin A/C (loading control) were measured to confirm the overexpression of CUX1 (B). Type I collagen, from cell supernatant, was also screened (middle panel, B). Densitometry was carried out to quantify the Western blot findings (B). The activity of the COL1A2 promoter was measured in the transfectant CUX1 lines α, β, and γ and in cells transfected with an EV using a specially designed DNA construct that carries the untranslated regulatory sequences of the COL1A2 gene linked to LacZ to allow measurement of promoter activity. COL1A2 promoter activity was also measured in conditions where the promoter of COL1A2 was genetically modified by introducing a point mutation at –80 base pairs (see A for sequence information of mutation), termed CBF mutant construct (C). Two double-stranded DNA probes, shown in (A), were used in EMSA experiments. CUX1 was found bound to the promoter of COL1A2 when using probe 1 (arrows) in TK173 cells overexpressing CUX1. CUX1 binding was removed with specific CUX1 oligonucleotide competition (lane 4) but was unaffected with control competition with CBF, C/EBP, SP1, and NF-κB (lanes 5–8) (D). CUX1, when overexpressed, was also found bound to an alternative site of the COL1A2 promoter, using probe 2. This binding was partially competed with CUX1 competition (lane 3, arrowhead) and partially with CBF (lane 4, asterisk). Control SP1 and C/EBP competition had no effect (lanes 5 and 6) (E).

FIGURE 3:

TGF-β suppresses type I collagen via induction of CUX1 at high doses. We measured the mRNA expression of CUX1 and COL1A2 by qPCR in normal collagen-producing fibroblastic cells (TK173) stimulated with increasing concentrations of TGF-β (1, 2, 5, and 10 ng/ml). CUX1 mRNA increased in response to TGF-β in a dose-dependent manner, reaching maximum stimulation at 10 ng/ml of TGFβ (A). COL1A2 mRNA increased in response to TGF-β (up to 5 ng/ml), but addition of TGF-β at high doses (10 ng/ml) had no stimulatory effects (A). These results were validated by measuring protein expression of CUX1 and collagen using Western blotting and by densitometry to quantify the findings (B). COL1A2 promoter activity was then measured in cells overexpressing either the p200 or the p75 isoforms of CUX1 and compared with EV-transfected kidney cells. TGF-β increased promoter activity of COL1A2 in EV cells at 2 ng/ml but consistently had no effect at 10 ng/ml. Overexpression of the CUX1 isoforms resulted in abolishing the TGF-β effects in promoting COL1A2 promoter activation (C). Using immunofluorescence, analysis of CUX1 intensity and localization was investigated in response to stimulation with 2 or 10 ng/ml of TGF-β and compared with vehicle-treated cells (left panel, D). Quantification of nuclear localization of CUX1 and pSMAD3 in response to TGF-β treatment was quantified (right panel, D). CUX1 was knocked down using two specific siRNA oligonucleotides, and efficiency of siRNA silencing was tested by qPCR in TK173 cells stimulated with TGF-β (E). Protein levels of type I collagen, CUX1, and β-actin were studied in TGF-β–stimulated cells with and without specific siRNA (F).

FIGURE 4:

TGF-β drives a mechanistic switch from Smad3- to p38-dependent activation of CUX1. The ability of TGF-β to increase binding of CUX1 to the COL1A2 promoter was investigated. TK173 stimulated with low (2 ng/ml) and high (10 ng/ml) TGF-β concentration were subjected to EMSA. CUX1 was found bound to the COL1A2 promoter only at the high TGF-β concentrations (A). The localization and intensity of the phosphorylated form of Smad3 was studied in TK173 cells in response to TGF-β stimulation. Low TGF-β (2 ng/ml) caused an induction of phospho-Smad3 that was primarily nuclear; however, high TGF-β (10 ng/ml) failed to trigger nuclear localization of phopsho-Smad3 (B). Then Smad 2 or 3 was selectively knocked down using specific siRNA oligonucleotides, and the effects that the knockdown had on CUX1 induction in response to TGF-β were studied. Smad2 and 3 knockdown significantly reduced TGF-β–induced CUX1 expression at the lower range (5 ng/ml) but had no effect at the higher concentrations of TGF-β (C). We then investigated the relative contribution of MAP kinases using pharmacological inhibition of JNK and p38. JNK and p38 inhibitors had no effect at all. P38 reduced TGF-β-induced CUX1 expression selectively at the high concentrations of TGF-β (10 ng/ml) but had no effect at the low concentrations (D).

FIGURE 5:

CUX1 is reduced in progressive interstitial fibrosis in vivo. AA was injected intraperitoneally into male C57BL6 mice to induce kidney fibrosis, and animals were killed at 0, 28, and 56 dpi. Their kidneys were collected, and histological sections were stained with picrosirius red to detect fibrillar collagen deposition (A). CUX1 mRNA was measured in whole kidney lysates from the AA model (B). Protein levels of CUX1 and type I collagen were also studied in the model (C). CUX1 was then overexpressed in fibroblasts derived from fibrotic regions of human kidney (TK188), where CUX1 expression is low. CUX1 mRNA and COL1A2 promoter activity were measured (D). CUX1, COL1A2, and β-actin protein expression was then investigated in fibrotic fibroblasts (E). The occupancy of the CUX1 site was studied in whole kidney lysates from control mice or 28 and 56 dpi. Using EMSA, we showed that CUX1 binds to the COL1A2 promoter in control animals on day 0, and this binding is diminished by 28 dpi and by day 56 of established fibrosis (F).