Cytokine-Like 1 Regulates Cardiac Fibrosis via Modulation of TGF-β Signaling

Abstract

Cytokine-like 1 (Cytl1) is a secreted protein that is involved in diverse biological processes. A comparative modeling study indicated that Cytl1 is structurally and functionally similar to monocyte chemoattractant protein 1 (MCP-1). As MCP-1 plays an important role in cardiac fibrosis (CF) and heart failure (HF), we investigated the role of Cytl1 in a mouse model of CF and HF. Cytl1 was upregulated in the failing mouse heart. Pressure overload-induced CF was significantly attenuated in cytl1 knock-out (KO) mice compared to that from wild-type (WT) mice. By contrast, adeno-associated virus (AAV)-mediated overexpression of cytl1 alone led to the development of CF in vivo. The endothelial-mesenchymal transition (EndMT) and the transdifferentiation of fibroblasts (FBs) to myofibroblasts (MFBs) have been suggested to contribute considerably to CF. Adenovirus-mediated overexpression of cytl1 was sufficient to induce these two critical CF-related processes in vitro, which were completely abrogated by co-treatment with SB-431542, an antagonist of TGF-β receptor 1. Cytl1 induced the expression of TGF-β2 both in vivo and in vitro. Antagonizing the receptor for MCP-1, C-C chemokine receptor type 2 (CCR2), with CAS 445479-97-0 did not block the pro-fibrotic activity of Cytl1 in vitro. Collectively, our data suggest that Cytl1 plays an essential role in CF likely through activating the TGF-β-SMAD signaling pathway. Although the receptor for Cyt1l remains to be identified, Cytl1 provides a novel platform for the development of anti-CF therapies.

Fig 1. Cytl1 is upregulated under pathological conditions.

Three groups of WT mice were subjected to TAC for 6 wks (TAC), ligation of coronary artery for 4 wks (MI), or ligation of coronary artery for 30 min followed by reperfusion for 24 hrs (I/R). Hearts were harvested and qRT-PCR was performed to determine the transcript levels of Cytl1. n = 3 for sham, n = 3 for TAC, n = 4 for MI, n = 4 for I/R. *p < 0.05, **p < 0.01.

Fig 2. CF is attenuated in cytl1 KO mice.

WT and cytl1 KO mice were subjected to TAC for 6 wks and the extent of fibrosis in the heart was analyzed. (A) Picrosirius staining of heart cross-sections from WT and cytl1 KO mice subjected to TAC. Fibrotic areas in the interstitial and perivascular areas were quantified using MetaMorph software (right panels). (B) Quantification of the mRNA levels of several fibrotic markers (TGF-β2, collagen 1 and TNF-α) by qRT-PCR. (C) Activation of the TGF-β signaling pathway was investigated by western blotting. GAPDH served as the loading control. n = 3–5 for each experimental group. *p < 0.05, **p < 0.01.

Fig 3. AAV-mediated overexpression of Cytl1 induces CF.

Control virus or AAV-Cytl1 (5 × 10¹⁰ viral genome) was injected into the tail vein of WT mice, and the phenotype of the heart was examined after 8 wks. (A) Picrosirius staining of heart cross-sections from WT mice injected with control virus or AAV-Cytl. Fibrotic areas in the interstitial and perivascular areas were quantified using MetaMorph software (right panels). (B) Quantification of the mRNA levels of several fibrotic markers (TGF-β2, collagen 1 and TNF-α) by qRT-PCR. (C) Activation of the TGF-β signaling pathway was investigated by western blotting. GAPDH served as the loading control. n = 3–5 for each experimental group. *p < 0.05, **p < 0.01.

Fig 4. Cytl1 induces EndMT.

HCAECs were treated with TGF-β2 (10 ng/ml) or Ad-Cytl1 (50 moi) for 48 h. In selected experiments, the cells were pretreated with the TGF-β receptor 1 antagonist SB431542 (10 μM). (A) The cells were immunostained with antibodies against the endothelial marker CD31 and the MFB marker α-SMA. (B) Lysates of HCAECs infected with Ad-Cytl1 were analyzed by western blotting. CD31 and VE-cadherin served as endothelial cell markers, and vimentin served as the MFB marker. (C) Activation of the TGF-β signaling pathway was investigated by western blotting. GAPDH served as the loading control. (D) Quantification of the mRNA levels of several fibrotic markers (TGF-β2, Cytl1, collagen 1 and TNF-α) by qRT-PCR. n = 3–5 for each experimental group. *p < 0.05, **p < 0.01.

Fig 5. Cytl1 induces transdifferentiation of FBs to MFBs.

Adult primary cardiac FBs were treated with TGF-β2 (10 ng/ml) or Ad-Cytl1 (50 moi) for 48 h. In selected experiments, the cells were pretreated with SB431542 (10 μM). (A) The cells were immunostained with an antibody against the MFB marker α-SMA. (B) Activation of the TGF-β signaling pathway in primary cardiac FBs infected with Ad-Cytl was investigated by western blotting. GAPDH served as the loading control. (C) Quantification of the mRNA levels of TGF-β2, Cytl1, the MFB marker collagen 1 and α-SMA by qRT-PCR. n = 3–5 for each experimental group. *p < 0.05, **p < 0.01.

Fig 6. Cytl1 functions independently of CCR2.

Adult primary cardiac FBs were treated with CCL2 (20 ng/ml) or Ad-Cytl1 (50 moi) for 48 h. In selected experiments, the cells were pretreated with the CCR2 antagonist CAS445679-97-0 (6 nM). (A) The cells were immunostained with an antibody against α-SMA. (B) Quantification of the mRNA levels of TGF-β2, Cytl1, collagen 1 and α-SMA by qRT-PCR. n = 3–5 for each experimental group. *p < 0.05, **p < 0.01.