Hedgehog/GLI Signaling Activates Suppressor of Cytokine Signaling 1 (<i>SOCS1</i>) in Epidermal and Neural Tumor Cells

<div><p>Sustained hedgehog (Hh) signaling mediated by the GLI transcription factors is implicated in many types of cancer. Identification of Hh/GLI target genes modulating the activity of other pathways involved in tumor development promise to open new ways for better understanding of tumor development and maintenance. Here we show that SOCS1 is a direct target of Hh/GLI signaling in human keratinocytes and medulloblastoma cells. SOCS1 is a potent inhibitor of interferon gamma (IFN-y)/STAT1 signaling. IFN-у/STAT1 signaling can induce cell cycle arrest, apoptosis and anti-tumor immunity. The transcription factors GLI1 and GLI2 activate the SOCS1 promoter, which contains five putative GLI binding sites, and GLI2 binding to the promoter was shown by chromatin immunoprecipitation. Consistent with a role of GLI in SOCS1 regulation, STAT1 phosphorylation is reduced in cells with active Hh/GLI signaling and IFN-у/STAT1 target gene activation is decreased. Furthermore, IFN-у signaling is restored by shRNA mediated knock down of SOCS1. Here, we identify SOCS1 as a novel Hh/GLI target gene, indicating a negative role of Hh/GLI pathway in IFN-y/STAT1 signaling.</p> </div

SOCS1 knock down restores IFN-у/STAT1 signaling in cells with activated Hh signaling.

<p><b>A</b>) Western blot analysis of SOCS1, GLI2, and ACTB in GLI2act-HaCaT cells transduced with two shRNAs directed against human SOCS1 (shSOCS1_1, shSOCS1_2) and control shRNA (shCTRL) expressing GLI2 for the time indicated (left panel). SOCS1 mRNA levels were also analyzed by qRT-PCR in cells expressing GLI2 for 48h. <b>B</b>) qRT-PCR of IFN-у target genes (CXCL10, CDKN1A, and ICAM1) measured in GLI2act-HaCaT cells after expressing GLI2 for 48h (+ DOX) with subsequent exposure to 1ng/ml recombinant IFN-у for 6h. <b>C</b>) qRT-PCR of IFN-у target gene activation (HLA-DRA, ICAM1, IFIT1, TRIM22 and IRF1) in DAOY cell lines stably expressing either shSOCS1_1 and shSOCS1_2 or unspecific control shRNA (shCTRL). Cells were pretreated with 200 nM SAG for 120h to activate the Hh pathway and subsequently incubated with 1ng/ml recombinant IFN-у for 6h. mRNA levels are shown as ratio of IFN-у treated to untreated samples. Data are given as mean ± SD of biological triplicates.</p

Reduced anchorage-independent growth of Hh-active DAOY cells in response to SOCS1 knock down.

<p><b>A</b>) SOCS1 mRNA levels were analyzed by qRT-PCR in DAOY and GLI2act-DAOY (24h GLI2act expression), showing efficient SOCS1 knock down. Black bar: shCTRL, light grey bar: shSOCS1_1, dark grey bar: shSOCS1_2. <b>B</b>) Anchorage independent growth of DAOY cells (top) or GLI2act-DAOY (middle: uninduced GLI2act DAOY (-Dox), bottom: induced GLI2act DAOY (+Dox)) expressing shCTRL, shSOCS1_1 or shSOCS1_2. Number and size of colonies are enhanced in response to GLI2act expression and reduced in presence of SOCS1 knock down. <b>C</b>) Quantification of assays shown in B). The large diagram shows the total number of all colonies, the small diagram refers to the number of colonies with a diameter larger than 200 µm. rel. no. CFU relative number of colony forming units. Black bar: shCTRL, light grey bar: shSOCS1_1, dark grey bar: shSOCS1_2. Error bars represent ± SD of biological quadruplicates, * P < 0.05, ** P < 0.01.</p

Model showing negative cross talk of Hh signaling with the IFN-у /STAT1 signaling cascade.

<p>Activation of Hh/GLI signaling enhances SOCS1 transcription, thereby downregulating IFN-у signal transduction by circumventing STAT1 phosphorylation and dimerization.</p

SOCS1 is a direct transcriptional target of GLI.

<p><b>A</b>) Graphical overview of the cloned SOCS1 promoter region containing 5 putative GLI binding sites. Numbers refer to the transcription start site (RefSeq NM_003745.1). Sequences of putative GLI binding sites are listed on the right. Bases differing from GLI consensus sequence are underlined. <b>B</b>) Luciferase assay of a 1822bp fragment of the human SOCS1 promoter (SOCS1prom, see A) and deletion construct SOCS1promdel (see A). HaCaT cells were co-transfected with SOCS1 reporter and GLI expression plasmids as indicated. +/-SD refers to quadruplicate samples. <b>C</b>) Chromatin immunoprecipitation shows specific binding of GLI2 to the SOCS1 promoter. Chromatin isolated from doxycycline (DOX) treated GLI2act-HaCaT cells was precipitated with either GLI2 specific antibody (αGLI2) or unspecific (normal IgG) antibody as control. Two fragments (F1 and F2) spanning BS2, BS3, and BS4 or BS5 were amplified from the SOCS1 promoter by PCR. As positive control a 148-bp fragment (PTCHprom) from the PTCH promoter was used [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075317#B49" target="_blank">49</a>] and a 284-bp fragment (RPLP0prom) from the human RPLP0 promoter served as negative control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075317#B70" target="_blank">70</a>]. * P < 0.001.</p

SOCS1 is expressed in basal cell carcinoma (BCC).

<p><b>A</b>) mRNA levels of hedgehog target genes GLI1 and SOCS1 in biopsies of human BCCs (n=7) and normal human skin samples (NS) (n=3) by qRT-PCR. Data were normalized to RPLP0 and represent mean values of all tested BCC and NS samples. Fold change refers to the ratio of BCC to NS. <b>B</b>) Immunostaining of sections of human BCCs (left) and normal skin (NS, right) with SOCS1 antibody.</p

Hh/GLI signaling induces SOCS1 expression.

<p><b>A</b>) and <b>B</b>) qRT–PCR of SOCS1 (black bars) and PTCH (grey bars) mRNA levels in HaCaT keratinocytes expressing GLI1 (GLI1-HaCaT) (A) or GLI2act (GLI2act-HaCaT) (B) under doxycycline (DOX) control for the times indicated. <b>C</b>) Western blot of SOCS1 and GLI2act protein level in DOX treated and untreated GLI2act-HaCaT cells. Beta-actin (ACTB) was used as loading control. <b>D</b>) and <b>E</b>) qRT–PCR of SOCS1 and PTCH expression in the keratinocyte cell line N/TERT-1 retrovirally transduced with GLI2act (pLL-GLI2act) or enhanced green fluorescent protein (EGFP) (pLL) as control. Cells were assayed 48h post infection. Fold change refers to mRNA ratio of GLI2act to EGFP expressing cells. <b>F</b>) qRT-PCR of SOCS1 mRNA in DAOY cells retrovirally transduced with EGFP tagged GLI1 (pLL-GLI1), GLI2act (pLL-GLI2act) or EGFP (pLL). Lower panel: Western blot of GLI1 and GLI2act transgene expression using EGFP antibody. <b>G</b>) DAOY cells were treated with Hh pathway agonist SAG alone or in combination with the antagonist cyclopamine (CYC) for 120h and analyzed for the expression of SOCS1 and PTCH by qRT-PCR. Controls were treated with DMSO only. Lower panel: Activation of the Hh pathway was monitored by Western blot using a GLI1 specific antibody. Error bars represent ± SD of biological triplicates. * unspecific signal.</p

Immunohistochemical Detection of CTGF in the Human Eye

<p><i>Purpose/Aim of the study</i>: Connective tissue growth factor (CTGF) is a key player in the control of extracellular matrix remodeling, fibrosis, and angiogenesis. It is also involved in the modification of the trabecular meshwork, thus potentially modulating outflow facility and intraocular pressure (IOP). As a consequence, CTGF might be relevant for the development of elevated IOP, a major risk factor in glaucoma-pathogenesis. While comprehensive information on the origins of CTGF in the human eye is not available, the goal of this study is to identify ocular sources of CTGF using morphological methods.</p> <p><i>Materials and Methods</i>: Human donor eyes were prepared for immunohistochemical analysis of CTGF, α-smooth muscle-actin (ASMA), and CD31. Confocal laser scanning microscopy was used for documentation.</p> <p><i>Results</i>: In the cornea, CTGF-immunoreactivity (CTGF-IR) was detected in the epithelium, mainly in basal layers, stromal keratinocytes, and endothelial cells. Adjacent conjunctiva showed also CTGF-IR in epithelial cells. In the iris, both, the sphincter and dilator muscles displayed CGTF-IR, as did iris and ciliary body vessels, deriving at this location from the vascular endothelium, as detected with CD31, but not from vascular smooth muscle cells, as detected with ASMA. In the ciliary body, CTGF-IR was detected in smooth-muscle cells of the ciliary muscle and further in the non-pigmented epithelium. In the retina, CTGF-IR was detected in the NFL and weakly in the IPL/OPL. In the choroid, the choriocapillaris and blood vessels displayed CTGF-IR. Further, few cells in the optic nerve head and the lamina cribrosa were CTGF-positive.</p> <p><i>Conclusion</i>: CTGF was detected in various structures of the human eye. Since CTGF has been also described in aqueous humor, the identified structures might be the sources of CTGF in the aqueous humor. By means of aqueous flow, CTGF is transported into the trabecular meshwork, where it could change outflow facility and therefore affecting IOP homeostasis.</p