KLF2 attenuates bleomycin-induced pulmonary fibrosis and inflammation with regulation of AP-1

Pulmonary fibrosis (PF) is a chronic, fibrosing interstitial pneumonia and devastating disease. Here we investigated the potential roles of Kruppel-like factor 2 (KLF2) on pulmonary fibrosis and inflammation response. A mouse model of pulmonary fibrosis was established by intratracheal injection of bleomycin (BLM). The mRNA and protein levels of KLF2 were assayed by RT-PCR and Western blotting respectively. The extent of lung fibrosis was determined using hematoxylin and eosin (HE) staining and Masson's trichrome staining, and the hydroxyproline content was quantified. RT-PCR was used to evaluate the mRNA expression of collagen type 1a1 (col1a1), col3a1, α-SMA, TNF-α, IL-1β and IL-6. The concentrations of TNF-α, IL-1β, and IL-6 in bronchoalveolar lavage fluid (BALF) and lung tissue were examined by ELISA. Also, the effects of KLF2 on activator protein-1 (AP-1) were evaluated by measuring the c-Jun and c-Fos protein levels. We found that KLF2 was remarkably downregulated in BLM-treated rats, both in mRNA and protein levels. Additionally, overexpression of KLF2 attenuated the destruction of the alveolar space and pulmonary interstitial collagen hyperplasia, and deposition reduced the expression of col1a1, col3a1, and α-SMA, and blocked the production of TNF-α, IL-1β, and IL-6 in BALF and lung tissue in vivo. Moreover, adenoviral transduction of KLF2 inhibited TGF-β1-induced expression of col1a1, col3a1, and α-SMA in vitro. Mechanically, BLM up-regulated c-Jun and c-Fos expression, which was impeded by KLF2 overexpression. Taken together, our data indicate that KLF2 attenuates pulmonary fibrosis and inflammation, possibly through the regulation of AP-1.     

TRPC1 protein channel is major regulator of epidermal growth factor receptor signaling

TRP channels have been associated with cell proliferation and aggressiveness in several cancers. In particular, TRPC1 regulates cell proliferation and motility, two processes underlying cancer progression. We and others have described the mechanisms of TRPC1-dependent cell migration. However, the involvement of TRPC1 in cell proliferation remains unexplained. In this study, we show that siRNA-mediated TRPC1 depletion in non small cell lung carcinoma cell lines induced G(0)/G(1) cell cycle arrest resulting in dramatic decrease in cell growth. The expression of cyclins D1 and D3 was reduced after TRPC1 knockdown, pointing out the role of TRPC1 in G(1)/S transition. This was associated with a decreased phosphorylation and activation of EGFR and with a subsequent disruption of PI3K/Akt and MAPK downstream pathways. Stimulation of EGFR by its natural ligand, EGF, induced Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry through TRPC1. Ca(2+) entry through TRPC1 conversely activated EGFR, suggesting that TRPC1 is a component of a Ca(2+)-dependent amplification of EGF-dependent cell proliferation.     

Polycomb Antagonizes p300/CREB-binding Protein-associated Factor to Silence FOXP3 in a Kruppel-like Factor-dependent Manner

Inducible gene expression underlies the epigenetically inherited differentiation program of most immune cells. We report that the promoter of the FOXP3 gene possesses two distinct functional states: an “off state” mediated by the polycomb histone methyltransferase complex and a histone acetyltransferase-dependent “on state.” Regulating these states is the presence of a Kruppel-like factor (KLF)-containing Polycomb response element. In the KLF10^−/− mouse, the FOXP3 promoter is epigenetically silenced by EZH2 (Enhancer of Zeste 2)-mediated trimethylation of Histone 3 K27; thus, impaired FOXP3 induction and inappropriate adaptive T regulatory cell differentiation results in vitro and in vivo. The epigenetic transmittance of adaptive T regulatory cell deficiency is demonstrated throughout more than 40 generations of mice. These results provide insight into chromatin remodeling events key to phenotypic features of distinct T cell populations.     

Proliferating cell nuclear antigen-dependent rapid recruitment of Cdt1 and CRL4Cdt2 at DNA-damaged sites after UV irradiation in HeLa cells

The licensing factor Cdt1 is degraded by CRL4(Cdt2) ubiquitin ligase dependent on proliferating cell nuclear antigen (PCNA) during S phase and when DNA damage is induced in G(1) phase. Association of both Cdt2 and PCNA with chromatin was observed in S phase and after UV irradiation. Here we used a micropore UV irradiation assay to examine Cdt2 accumulation at cyclobutane pyrimidine dimer-containing DNA-damaged sites in the process of Cdt1 degradation in HeLa cells. Cdt2, present in the nucleus throughout the cell cycle, accumulated rapidly at damaged DNA sites during G(1) phase. The recruitment of Cdt2 is dependent on prior PCNA chromatin binding because Cdt2 association was prevented when PCNA was silenced. Cdt1 was also recruited to damaged sites soon after UV irradiation through its PIP-box. As Cdt1 was degraded, the Cdt2 signal at damaged sites was reduced, but PCNA, cyclobutane pyrimidine dimer, and XPA (xeroderma pigmentosum, complementation group A) signals remained at the same levels. These findings suggest that Cdt1 degradation following UV irradiation occurs rapidly at damaged sites due to PCNA chromatin loading and the recruitment of Cdt1 and CRL4(Cdt2), before DNA damage repair is completed.     

Prolylcarboxypeptidase regulates proliferation, autophagy, and resistance to 4-hydroxytamoxifen-induced cytotoxicity in estrogen receptor-positive breast cancer cells

Endocrine therapy with tamoxifen (TAM) significantly improves outcomes for patients with estrogen receptor-positive breast cancer. However, intrinsic (de novo) or acquired resistance to TAM occurs in a significant proportion of treated patients. To identify genes involved in resistance to TAM, we introduced full-length cDNA expression library into estrogen receptor-positive MCF7 cells and exposed them to a cytotoxic dose of 4-hydroxytamoxifen (4OHTAM). Four different library inserts were isolated from surviving clones. Re-introduction of the genes individually into naive MCF7 cells made them resistant to 4OHTAM. Cells overexpressing these genes had an increase in acidic autophagic vacuoles induced by 4OHTAM, suggesting their role in autophagy. One of them, prolylcarboxypeptidase (PRCP), was investigated further. Overexpression of PRCP increased cell proliferation, boosted several established markers of autophagy, including expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA in an ER-α dependent manner, and increased resistance to 4OHTAM. Conversely, knockdown of endogenous PRCP in MCF7 cells increased cell sensitivity to 4OHTAM and at the same time decreased cell proliferation and expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA. Inhibition of enzymatic activity of PRCP enhanced 4OHTAM-induced cytotoxicity in MCF7 cells. Cells with acquired resistance to 4OHTAM exhibited increased PRCP activity, although inhibition of PRCP prevented development of 4OHTAM resistance in parental MCF7 cells and restored response to 4OHTAM in MCF7 cells with acquired resistance to 4OHTAM. Thus, we have for the first time identified PRCP as a resistance factor for 4OHTAM resistance in estrogen receptor-positive breast cancer cells.     

A novel gene STYK1/NOK is upregulated in estrogen receptor-alpha negative estrogen receptor-beta positive breast cancer cells following estrogen treatment

The human STYK1/NOK protein is approximately 30–35% similar to mouse fibroblast growth factor receptor 3 and a kinase homologue in D. melanogaster in the tyrosine protein kinase region. STYK1/NOK was identified as being up regulated in MDA-MB-231, an estrogen receptor-alpha negative breast cancer cell line, following 12 h of estrogen treatment at 1 × 10⁻⁹ M. On further investigation of STYK1/NOK in estrogen treated cell line MDA-MB-231, STYK1/NOK was up regulated at 6 h post treatment when compared to untreated cells. We also investigated the expression levels of STYK1/NOK in other breast cancer cell lines MCF-7, MDA-MB-231, BT-549, and MDA-MB-435S using QRT-PCR. In addition, the analysis of message accumulation was increased with other synthetic estrogen response modifiers. We propose that the regulation of STYK1/NOK is achieved independent of ERα and suggests further investigation to the relevance of this kinase in breast cancer progression.     

The breast cancer cells response to chronic hypoxia involves the opposite regulation of NF-kB and estrogen receptor signaling

The progression of cancer is associated with tumor's ability to outgrow the existing vasculature resulting in chronic hypoxic pressure, however the molecular mechanism of cancer cell response to chronic hypoxia is poorly understood. In this study we have analyzed the reorganization of estrogen receptor (ER) signaling in breast cancer cells under chronic hypoxia and examined the role of interrelations between ER and NF-kB signaling in cell adaptation to hypoxia. Using long-term culturing of MCF-7 breast cancer cells in hypoxia-mimetic conditions (cobalt chloride) we have established a hypoxia-tolerant subline characterized by HIF-1 hyperexpression that retained the tolerance to hypoxia even when the cells were returned to normoxic conditions.The hypoxia-tolerant cells were characterized by non-affected ER signaling, irreversible suppression of NF-kB activity, and increased sensitivity to cytokine-induced apoptosis. Estradiol treatment suppressed the NF-kB activity in both parent and hypoxia-tolerant MCF-7 cells. In contrast to MCF-7 cells, the exposure of estrogen-independent MCF-7/T2 subline to chronic hypoxia was not accompanied by noticeable changes in NF-kB activity or cell sensitivity to cytokines. Taken together, the results presented demonstrate the importance of interrelations between ER and NF-kB signaling in the response of estrogen-dependent breast cancer cells to chronic hypoxia.     

Distinctive molecular signaling in triple-negative breast cancer cell death triggered by hexadecylphosphocholine (miltefosine)

This study describes the molecular signaling involved in the different cell death modes of triple-negative breast cancer cells induced by hexadecylphosphocholine (HePC/miltefosine), a clinically relevant anticancer alkylphosphocholine. We found that the HePC treatment triggers cell-type-dependent apoptotic and non-apoptotic cell death processes. Moreover, the expression level of the apoptosis activator Fas, and Fas/Fas ligand signaling capacity are not attributing factors for the preference toward apoptosis. Using Fas siRNA and overexpression approaches we establish that Fas is not a pro-apoptotic factor but a contributor to cell protection in HePC-apoptosis-sensitive cells. The insight in the multi-modal anticancer capability of HePC in triple-negative breast cancer cells may facilitate the targeted design of therapeutic strategies against triple-negative breast cancers.