Pulmonary fibrosis is a progressive inflammatory disease with high mortality and limited therapeutic options. Previous genetic and immunologic investigations suggest common intersections between idiopathic pulmonary fibrosis (IPF), sarcoidosis, and murine models of pulmonary fibrosis. To identify immune responses that precede collagen deposition, we conducted molecular, immunohistochemical, and flow cytometric analysis of human and murine specimens. Immunohistochemistry revealed programmed cell death-1 (PD-1) up-regulation on IPF lymphocytes. PD-1+CD4+ T cells with reduced proliferative capacity and increased transforming growth factor–β (TGF-β)/interleukin-17A (IL-17A) expression were detected in IPF, sarcoidosis, and bleomycin CD4+T Cells. PD-1+ T helper 17 cells are the predominant CD4+T cell subset expressing TGF-β. Coculture of PD-1+CD4+ T cells with human lung fibroblasts induced collagen-1 production. Strikingly, ex vivo PD-1 pathway blockade resulted in reductions in TGF-β and IL-17A expression from CD4+ T cells, with concomitant declines in collagen-1 production from fibroblasts. Molecular analysis demonstrated PD-1 regulation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Chemical blockade of STAT3, using the inhibitor STATTIC, inhibited collagen-1 production. Both bleomycin administration to PD-1 null mice or use of antibody against programmed cell death ligand 1 (PD-L1) demonstrated significantly reduced fibrosis compared to controls. This work identifies a critical, previously unrecognized role for PD-1+CD4+ T cells in pulmonary fibrosis, supporting the use of readily available therapeutics that directly address interstitial lung disease pathophysiology.

Elevated activity of Src, the first characterized protein-tyrosine kinase, is associated with progression of many human cancers, and Src has attracted interest as a therapeutic target. Src is known to act in various receptor signaling systems to impact cell behavior, yet it remains likely that the spectrum of Src protein substrates relevant to cancer is incompletely understood. To better understand the cellular impact of deregulated Src kinase activity, we extensively applied a mass spectrometry shotgun phosphotyrosine (pTyr) proteomics strategy to obtain global pTyr profiles of Src-transformed mouse fibroblasts as well as their nontransformed counterparts. A total of 867 peptides representing 563 distinct pTyr sites on 374 different proteins were identified from the Src-transformed cells, while 514 peptides representing 275 pTyr sites on 167 proteins were identified from nontransformed cells. Distinct characteristics of the two profiles were revealed by spectral counting, indicative of pTyr site relative abundance, and by complementary quantitative analysis using stable isotope labeling with amino acids in cell culture (SILAC). While both pTyr profiles are replete with sites on signaling and adhesion/cytoskeletal regulatory proteins, the Src-transformed profile is more diverse with enrichment in sites on metabolic enzymes and RNA and protein synthesis and processing machinery. Forty-three pTyr sites (32 proteins) are predicted as major biologically relevant Src targets on the basis of frequent identification in both cell populations. This select group, of particular interest as diagnostic biomarkers, includes well-established Src sites on signaling/adhesion/cytoskeletal proteins, but also uncharacterized sites of potential relevance to the transformed cell phenotype.

Transcription of the catalytic subunit of telomerase (hTERT) in keratinocytes can be induced by human papillomavirus type 16 (HPV16) E6/E6AP ubiquitin ligase through degradation of the repressor, NFX1-91. Here, we demonstrate that NFX1-91 interacts with the corepressor complex mSin3A/histone deacetylase (HDAC) at the hTERT promoter. By degrading NFX1-91, E6/E6AP changes the chromatin structure at the hTERT promoter as indicated by enhanced acetylation of histones H3 and H4 as well as dimethylation of H3K4. Knockdown of NFX1-91 by short hairpin RNA (shRNA) mimics the effect of E6 and leads to acetylation of histones H3 and H4. Conversely, knockdown of E6AP by shRNA suppresses histone acetylation at the hTERT promoter. These data demonstrate that targeted degradation of NFX1-91 by E6/E6AP dissociates the mSin3A/ HDAC complex from the hTERT promoter and induces hTERT transcription.Maintenance of telomere length by telomerase is required for sustained cell proliferation, and activation of telomerase is a critical step during cellular immortalization and malignant transformation (7). Expression of the catalytic subunit of telomerase (hTERT) is rate limiting for telomerase activity (28,30,32); thus, understanding the regulation of hTERT transcription is critical to elucidate how cells acquire immortality. The hTERT promoter contains several transcription factorbinding sites, including c-Myc (15, 42, 45), Sp1 (18,37,43), USF (12), and ER81 (13). Several studies showed that hTERT expression is minimal or absent in most somatic cells through both known transcriptional repressors, such as Mad1, menin, and SIP1 (24), and other endogenous factors demonstrated by chromosome transfer experiments, such as a putative tumor suppressor on chromosome 3p (17). Distinct repression and activation mechanisms likely operate in different tissues. For example, a complex containing BRCA1 inhibits c-Myc-induced hTERT promoter activity in breast cancer cells (23), estrogen upregulates telomerase in both mammary and ovarian epithelial cells (3,29), and E2F-1 activates the hTERT promoter in some normal human somatic cells (44).The human papillomavirus type 16 (HPV16) E6 oncoprotein can induce telomerase activity in human primary keratinocytes through induction of hTERT expression (9,10,33,39). Studies on how E6 regulates hTERT have implicated E-box and X-box elements in the hTERT promoter (9, 10, 33, 40). c-Myc's role in directly enhancing transcription of hTERT has been demonstrated in several cell lines (15,21,45). However, the mechanism is still controversial and not well defined (9, 33, 39, 40). Although both E-box and c-Myc activities were found to be required for E6-mediated activation of hTERT (33,39,40), neither increased c-Myc protein level nor enhanced MycMax complex formation was detected with E6 expression (9,33,39,40).Recently, we identified NFX1-91 as a transcriptional repressor that binds to a consensus X-box element at the hTERT promoter, which overlaps with an E box (10). Two isoforms of NFX1 generated by alternative splicing...