Deficiency of the placenta- and yolk sac-specific tristetraprolin family member ZFP36L3 identifies likely mRNA targets and an unexpected link to placental iron metabolism.

The ZFP36L3 protein is a rodent-specific, placenta- and yolk sac-specific member of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins. These proteins bind to AU-rich elements in target mRNAs, and promote their deadenylation and decay. We addressed the hypotheses that the absence of ZFP36L3 would result in the accumulation of target transcripts in placenta and/or yolk sac, and that some of these would be important for female reproductive physiology and overall fecundity. Mice deficient in ZFP36L3 exhibited decreased neonatal survival rates, but no apparent morphological changes in the placenta or surviving offspring. We found Zfp36l3 to be paternally imprinted, with profound parent-of-origin effects on gene expression. The protein was highly expressed in the syncytiotrophoblast cells of the labyrinth layer of the placenta, and the epithelial cells of the yolk sac. RNA-Seq of placental mRNA from Zfp36l3 knockout (KO) mice revealed many significantly upregulated transcripts, whereas there were few changes in KO yolk sacs. Many of the upregulated placental transcripts exhibited decreased decay rates in differentiated trophoblast stem cells derived from KO blastocysts. Several dozen transcripts were deemed high probability targets of ZFP36L3; these include proteins known to be involved in trophoblast and placenta physiology. Type 1 transferrin receptor mRNA was unexpectedly decreased in KO placentas, despite an increase in its stability in KO stem cells. This receptor is crucial for placental iron uptake, and its decrease was accompanied by decreased iron stores in the KO fetus, suggesting that this intrauterine deficiency might have deleterious consequences in later life.

Instillation of hyaluronan reverses acid instillation injury to the mammalian blood gas barrier.

Acid (HCl) aspiration during anesthesia may lead to acute lung injury. There is no effective therapy. We hypothesized that HCl instilled intratracheally in C57BL/6 mice results in the formation of low-molecular weight hyaluronan (L-HA), which activates RhoA and Rho kinase (ROCK), causing airway hyperresponsiveness (AHR) and increased permeability. Furthermore, instillation of high-molecular weight hyaluronan (H-HA; Yabro) will reverse lung injury. We instilled HCl in C57BL/6 wild-type (WT), myeloperoxidase gene-deficient (MPO-/-) mice, and CD44 gene-deficient (CD44-/-) mice. WT mice were also instilled intranasally with H-HA (Yabro) at 1 and 23 h post-HCl. All measurements were performed at 1, 5, or 24 h post-HCl. Instillation of HCl in WT but not in CD44-/- resulted in increased inflammation, AHR, lung injury, and L-HA in the bronchoalveolar lavage fluid (BALF) 24 h post-HCl; L-HA levels and lung injury were significantly lower in HCl-instilled MPO-/- mice. Isolated perfused lungs of HCl instilled WT but not of CD44-/- mice had elevated values of the filtration coefficient ( Kf). Addition of L-HA on the apical surface of human primary bronchial epithelial cell monolayer decreased barrier resistance ( RT). H-HA significantly mitigated inflammation, AHR, and pulmonary vascular leakage at 24 h after HCl instillation and mitigated the increase of Kf and RT, as well as ROCK2 phosphorylation. Increased H- and L-HA levels were found in the BALF of mechanically ventilated patients but not in healthy volunteers. HCl instillation-induced lung injury is mediated by the L-HA-CD44-RhoA-ROCK2 signaling pathway, and H-HA is a potential novel therapeutic agent for acid aspiration-induced lung injury.

High molecular weight hyaluronan ameliorates allergic inflammation and airway hyperresponsiveness in the mouse.

Allergic asthma is a major cause of morbidity in both pediatric and adult patients. Recent research has highlighted the role of hyaluronan (HA), an extracellular matrix glycosaminoglycan, in asthma pathogenesis. Experimental allergic airway inflammation and clinical asthma are associated with an increase of shorter fragments of HA (sHA), which complex with inter-α-inhibitor heavy chains (HCs) and induce inflammation and airway hyperresponsiveness (AHR). Importantly, the effects of sHA can be antagonized by the physiological counterpart high molecular weight HA (HMWHA). We used a mouse model of house dust mite-induced allergic airway inflammation and demonstrated that instilled HMWHA ameliorated allergic airway inflammation and AHR, even when given after the establishment of allergic sensitization and after challenge exposures. Furthermore, instilled HMWHA reduced the development of HA-HC complexes and the activation of Rho-associated, coiled-coil containing protein kinase 2. We conclude that airway application of HMWHA is a potential treatment for allergic airway inflammation.

Targeted HAS2 Expression Lessens Airway Responsiveness in Chronic Murine Allergic Airway Disease.

Hyaluronan (HA), a major component of the extracellular matrix, is secreted by airway structural cells. Airway fibroblasts in allergic asthma secrete elevated levels of HA in association with increased HA synthase 2 (HAS2) expression. Thus, we hypothesized that HA accumulation in the airway wall may contribute to airway remodeling and hyperresponsiveness in allergic airways disease. To examine this hypothesis, transgenic mice in which the α-smooth muscle actin (α-SMA) promoter drives HAS2 expression were generated. Mixed male and female α-SMA-HAS2 mice (HAS2+ mice, n = 16; HAS2- mice, n = 13) were sensitized via intraperitoneal injection and then chronically challenged with aerosolized ovalbumin (OVA) for 6 weeks. To test airway responsiveness, increasing doses of methacholine were delivered intravenously and airway resistance was measured using the forced oscillation technique. HA, cytokines, and cell types were analyzed in bronchoalveolar lavage fluid, serum, and whole lung homogenates. Lung sections were stained using antibodies specific for HA-binding protein (HABP) and α-SMA, as well as Masson's trichrome stain. Staining of lung tissue demonstrated significantly increased peribronchial HA, α-SMA, and collagen deposition in OVA-challenged α-SMA-HAS2+ mice compared with α-SMA-HAS2- mice. Unexpectedly, OVA-challenged α-SMA-HAS2+ mice displayed significantly reduced airway responsiveness to methacholine compared with similarly treated α-SMA-HAS2- mice. The total numbers of inflammatory cell types in the bronchoalveolar lavage fluid did not differ significantly between OVA-challenged α-SMA-HAS2+ mice and α-SMA-HAS2- mice. We conclude that allergen-challenged mice that overexpress HAS2 in myofibroblasts and smooth muscle cells develop increased airway fibrosis, which lessens airway hyperresponsiveness to bronchoconstrictors.

A novel role for primary cilia in airway remodeling.

Primary cilia (PC) are solitary cellular organelles that play critical roles in development, homeostasis, and disease pathogenesis by modulating key signaling pathways such as Sonic Hedgehog and calcium flux. The antenna-like shape of PC enables them also to facilitate sensing of extracellular and mechanical stimuli into the cell, and a critical role for PC has been described for mesenchymal cells such as chondrocytes. However, nothing is known about the role of PC in airway smooth muscle cells (ASMCs) in the context of airway remodeling. We hypothesized that PC on ASMCs mediate cell contraction and are thus integral in the remodeling process. We found that PC are expressed on ASMCs in asthmatic lungs. Using pharmacological and genetic methods, we demonstrated that PC are necessary for ASMC contraction in a collagen gel three-dimensional model both in the absence of external stimulus and in response to the extracellular component hyaluronan. Mechanistically, we demonstrate that the effect of PC on ASMC contraction is, to a small extent, due to their effect on Sonic Hedgehog signaling and, to a larger extent, due to their effect on calcium influx and membrane depolarization. In conclusion, PC are necessary for the development of airway remodeling by mediating calcium flux and Sonic Hedgehog signaling.

The epidermal growth factor receptor decreases Stathmin 1 and triggers catagen entry in the mouse.

The epidermal growth factor receptor (EGFR) is necessary for normal involution of hair follicles after the growth phase of anagen, although the mechanisms through which it acts are not well understood. In this report, we used transcriptional profiling of microdissected hair follicles from mice with skin-targeted deletion of Egfr to investigate how EGFR activation triggers catagen. Immunofluorescence for phospho-EGFR in mouse skin revealed increased activation of EGFR in follicular keratinocytes at catagen onset. Consistent with other models of EGFR deficiency, mice with skin-targeted deletion of Egfr (Krt14-Cre(+) /Egfr(fl/fl) ) exhibited a delayed and asynchronous catagen entry. Transcriptional profiling at the time of normal catagen onset at post-natal day (P) 17 revealed increased expression of the mitotic regulator Rcc2 in hair follicles lacking EGFR. Rcc2 protein was strongly immunopositive in the nuclei of control follicular keratinocytes at P16 then rapidly decreased until it was undetectable between P18 and 21. In contrast, Rcc2 expression continued in Egfr mutant follicles throughout this period. Proliferation, measured by bromodeoxyuridine incorporation, was also significantly increased in Egfr mutant follicular keratinocytes compared to controls at P18-21. Similarly, Rcc2-regulated mitotic regulator Stathmin 1 was strikingly reduced in control but not Egfr mutant follicles between P17 and P19. Deletion of Stmn1, in turn, accelerated catagen entry associated with premature cessation of proliferation in the hair follicles. These data reveal EGFR suppression of mitotic regulators including Rcc2 and Stathmin 1 as a mechanism for catagen induction in mouse skin.

Hyaluronan mediates airway hyperresponsiveness in oxidative lung injury.

Chlorine (Cl2) inhalation induces severe oxidative lung injury and airway hyperresponsiveness (AHR) that lead to asthmalike symptoms. When inhaled, Cl2 reacts with epithelial lining fluid, forming by-products that damage hyaluronan, a constituent of the extracellular matrix, causing the release of low-molecular-weight fragments (L-HA, <300 kDa), which initiate a series of proinflammatory events. Cl2 (400 ppm, 30 min) exposure to mice caused an increase of L-HA and its binding partner, inter-α-trypsin-inhibitor (IαI), in the bronchoalveolar lavage fluid. Airway resistance following methacholine challenge was increased 24 h post-Cl2 exposure. Intratracheal administration of high-molecular-weight hyaluronan (H-HA) or an antibody against IαI post-Cl2 exposure decreased AHR. Exposure of human airway smooth muscle (HASM) cells to Cl2 (100 ppm, 10 min) or incubation with Cl2-exposed H-HA (which fragments it to L-HA) increased membrane potential depolarization, intracellular Ca(2+), and RhoA activation. Inhibition of RhoA, chelation of intracellular Ca(2+), blockade of cation channels, as well as postexposure addition of H-HA, reversed membrane depolarization in HASM cells. We propose a paradigm in which oxidative lung injury generates reactive species and L-HA that activates RhoA and Ca(2+) channels of airway smooth muscle cells, increasing their contractility and thus causing AHR.

Erbb2 up-regulation of ADAM12 expression accelerates skin cancer progression.

Solar ultraviolet (UV) radiation can cause severe damage to the skin and is the primary cause of most skin cancer. UV radiation causes DNA damage leading to mutations and also activates the Erbb2/HER2 receptor through indirect mechanisms involving reactive oxygen species. We hypothesized that Erbb2 activation accelerates the malignant progression of UV-induced skin cancer. Following the induction of benign squamous papillomas by UV exposure of v-ras(Ha) transgenic Tg.AC mice, mice were treated topically with the Erbb2 inhibitor AG825 and tumor progression monitored. AG825 treatment reduced tumor volume, increased tumor regression, and delayed the development of malignant squamous cell carcinoma (SCC). Progression to malignancy was associated with increased Erbb2 and ADAM12 (A Disintegin And Metalloproteinase 12) transcripts and protein, while inhibition of Erbb2 blocked the increase in ADAM12 message upon malignant progression. Similarly, human SCC and SCC cell lines had increased ADAM12 protein and transcripts when compared to normal controls. To determine whether Erbb2 up-regulation of ADAM12 contributed to malignant progression of skin cancer, Erbb2 expression was modulated in cultured SCC cells using forced over-expression or siRNA targeting, demonstrating up-regulation of ADAM12 by Erbb2. Furthermore, ADAM12 transfection or siRNA targeting revealed that ADAM12 increased both the migration and invasion of cutaneous SCC cells. Collectively, these results suggest Erbb2 up-regulation of ADAM12 as a novel mechanism contributing to the malignant progression of UV-induced skin cancer. Inhibition of Erbb2/HER2 reduced tumor burden, increased tumor regression, and delayed the progression of benign skin tumors to malignant SCC in UV-exposed mice. Inhibition of Erbb2 suppressed the increase in metalloproteinase ADAM12 expression in skin tumors, which in turn increased migration and tumor cell invasiveness.

A keratin 15 containing stem cell population from the hair follicle contributes to squamous papilloma development in the mouse.

The multistage model of nonmelanoma skin carcinogenesis has contributed significantly to our understanding of epithelial cancer in general. We used the Krt1-15CrePR1;R26R transgenic mouse to determine the contribution of keratin 15+ cells from the hair follicle to skin tumor development by following the labeled progeny of the keratin 15 expressing cells into papillomas. We present three novel observations. First, we found that keratin 15 expressing cells contribute to most of the papillomas by 20 weeks of promotion. Second, in contrast to the transient behavior of labeled keratin 15-derived progeny in skin wound healing, keratin 15 progeny persist in papillomas, and some malignancies for many months following transient induction of the reporter gene. Third, papillomas have surprising heterogeneity not only in their cellular composition, but also in their expression of the codon 61 signature Ha-ras mutation with approximately 30% of keratin 15-derived regions expressing the mutation. Together, these results demonstrate that keratin 15 expressing cells of the hair follicle contribute to cutaneous papillomas with long term persistence and a subset of which express the Ha-ras signature mutation characteristic of initiated cells.

Functional Pdgfra fibroblast heterogeneity in normal and fibrotic mouse lung.

Aberrant fibroblast function plays a key role in the pathogenesis of idiopathic pulmonary fibrosis, a devastating disease of unrelenting extracellular matrix deposition in response to lung injury. Platelet-derived growth factor α-positive (Pdgfra+) lipofibroblasts (LipoFBs) are essential for lung injury response and maintenance of a functional alveolar stem cell niche. Little is known about the effects of lung injury on LipoFB function. Here, we used single-cell RNA-Seq (scRNA-Seq) technology and PdgfraGFP lineage tracing to generate a transcriptomic profile of Pdgfra+ fibroblasts in normal and injured mouse lungs 14 days after bleomycin exposure, generating 11 unique transcriptomic clusters that segregated according to treatment. While normal and injured LipoFBs shared a common gene signature, injured LipoFBs acquired fibrogenic pathway activity with an attenuation of lipogenic pathways. In a 3D organoid model, injured Pdgfra+ fibroblast-supported organoids were morphologically distinct from those cultured with normal fibroblasts, and scRNA-Seq analysis suggested distinct transcriptomic changes in alveolar epithelia supported by injured Pdgfra+ fibroblasts. In summary, while LipoFBs in injured lung have not migrated from their niche and retain their lipogenic identity, they acquire a potentially reversible fibrogenic profile, which may alter the kinetics of epithelial regeneration and potentially contribute to dysregulated repair, leading to fibrosis.

Accelerated elimination of ultraviolet-induced DNA damage through apoptosis in CDC25A-deficient skin.

Cell division cycle 25A (CDC25A) is a dual-specificity phosphatase that removes inhibitory phosphates from cyclin-dependent kinases, allowing cell-cycle progression. Activation of cell-cycle checkpoints following DNA damage results in the degradation of CDC25A, leading to cell-cycle arrest. Ultraviolet (UV) irradiation, which causes most skin cancer, results in both DNA damage and CDC25A degradation. We hypothesized that ablation of CDC25A in the skin would increase cell-cycle arrest following UV irradiation, allowing for improved repair of DNA damage and decreased tumorigenesis. Cdc25a(fl/fl) /Krt14-Cre recombinase mice, with decreased CDC25A in the epithelium of the skin, were generated and exposed to UV. UV-induced DNA damage, in the form of cyclopyrimidine dimers and 8-oxo-deoxyguanosine adducts, was eliminated earlier from CDC25A-deficient epidermis. Surprisingly, loss of CDC25A did not alter epidermal proliferation or cell cycle after UV exposure. However, the UV-induced apoptotic response was prolonged in CDC25A-deficient skin. Double labeling of cleaved caspase-3 and the DNA damage marker γH2A.X revealed many of the apoptotic cells in UV-exposed Cdc25a mutant skin had high levels of DNA damage. Induction of skin tumors by UV irradiation of Cdc25a mutant and control mice on a skin tumor susceptible to v-ras(Ha) Tg.AC mouse background revealed UV-induced papillomas in Cdc25a mutants were significantly smaller than in controls in the first 6 weeks following UV exposure, although there was no difference in tumor multiplicity or incidence. Thus, deletion of Cdc25a increased apoptosis and accelerated the elimination of DNA damage following UV but did not substantially alter cell-cycle regulation or tumorigenesis.

A prolonged and exaggerated wound response with elevated ODC activity mimics early tumor development.

Induction of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, in ODC transgenic skin stimulates epidermal proliferation but not hyperplasia, activates underlying stromal cells and promotes skin tumorigenesis following a single subthreshold dose of a carcinogen. Because chronic wounds are a well-recognized risk factor for skin cancer, we investigated the response to a tissue remodeling event in normal skin that is abraded to remove only the epidermal layer in K6/ODC transgenic (follicular ODC expression) and in inducible ODCER transgenic mice (suprabasal ODC expression). When regenerative epidermal hyperplasia was resolved in normal littermates following abrasion, ODC transgenic mice exhibited progressive epidermal hyperplasia with formation of benign tumor growths and maintained an increased epidermal proliferation index and activation of translation-associated proteins at abrasion sites. The epidermal hyperplasia and tumor-like growth was accompanied by activation of underlying stromal cells and prolonged infiltration of inflammatory cells. Treatment with the anti-inflammatory agent dexamethasone did not reduce the high proliferative index in the regenerated epidermis but dramatically reduced the epidermal hyperplasia and prevented the wound-induced tumor growths in abraded ODCER skin. Treatment with α-difluoromethylornithine, a specific inhibitor of ODC activity, normalized the wound response in transgenic mice and decreased wound-induced inflammation if administered from the time of abrasion but not if initiated 4 days following abrasion. These results suggest a role for polyamines in prolonging wound-associated inflammation in addition to stimulating proliferation both of which are sufficient to sustain epidermal hyperplasia and benign tumor growth even in the absence of genetic damage.

A novel role for the T-box transcription factor Tbx1 as a negative regulator of tumor cell growth in mice.

The T-box transcription factor, Tbx1, an important regulatory gene in development, is highly expressed in hair follicle (HF) stem cells in adult mice. Because mouse models of skin carcinogenesis have demonstrated that HF stem cells are a carcinogen target population and contribute significantly to tumor development, we investigated whether Tbx1 plays a role in skin carcinogenesis. We first assessed Tbx1 expression levels in mouse skin tumors, and found down-regulation in all tumors examined. To study the effect of Tbx1 expression on growth and tumorigenic potential of carcinoma cells, we transfected mouse Tbx1 cDNA into a mouse spindle cell carcinoma cell line that did not express endogenous Tbx1. Following transfection, two cell lines expressing different levels of the Tbx1/V5 fusion protein were selected for further study. Intradermal injection of the cell lines into mice revealed that Tbx1 expression significantly suppressed tumor growth, albeit with no change in tumor morphology. In culture, ectopic Tbx1 expression resulted in decreased cell growth and reduced development into multilayered colonies, compared to control cells. Tbx1-transfectants exhibited a reduced proliferative rate compared to control cells, with fewer cells in S and G2/M phases. The Tbx1 transfectants developed significantly fewer colonies in soft agar, demonstrating loss of anchorage-independent growth. Taken together, our data show that ectopic expression of Tbx1 restored contact inhibition to the skin tumor cells, suggesting that this developmentally important transcription factor may have a novel dual role as a negative regulator of tumor growth. © 2011 Wiley Periodicals, Inc.

CD34 Antigen: Determination of Specific Sites of Phosphorylation In Vitro and In Vivo.

CD34, a type I transmembrane glycoprotein, is a surface antigen which is expressed on several cell types, including hematopoietic progenitors, endothelial cells, as well as mast cells. Recently, CD34 has been described as a marker for epidermal stem cells in mouse hair follicles, and is expressed in outer root sheath cells of the human hair follicle. Although the biological function and regulation of CD34 is not well understood, it is thought to be involved in cell adhesion as well as possibly having a role in signal transduction. In addition, CD34 was shown to be critical for skin tumor development in mice, although the exact mechanism remains unknown.Many proteins' functions and biological activities are regulated through post-translational modifications. The extracellular domain of CD34 is heavily glycosylated but the role of these glycans in CD34 function is unknown. Additionally, two sites of tyrosine phosphorylation have been reported on human CD34 and it is known that CD34 is phosphorylated, at least in part, by protein kinase C; however, the precise location of the sites of phosphorylation has not been reported. In an effort to identify specific phosphorylation sites in CD34 and delineate the possible role of protein kinase C, we undertook the identification of the in vitro sites of phosphorylation on the intracellular domain of mouse CD34 (aa 309-382) following PKC treatment. For this work, we are using a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. After which the in vivo sites of phosphorylation of full-length mouse CD34 expressed from HEK293F cells were determined. The observed in vivo sites of phosphorylation, however, are not consensus PKC sites, but our data indicate that one of these sites may possibly be phosphorylated by AKT2. These results suggest that other kinases, as well as PKC, may have important signaling functions in CD34.

Erbb2 suppresses DNA damage-induced checkpoint activation and UV-induced mouse skin tumorigenesis.

The Erbb2 receptor is activated by UV irradiation, the primary cause of non-melanoma skin cancer. We hypothesized that Erbb2 activation contributes to UV-induced skin tumorigenesis by suppressing cell cycle arrest. Consistent with this hypothesis, inhibition of Erbb2 in v-ras(Ha) transgenic mice before UV exposure resulted in both 56% fewer skin tumors and tumors that were 70% smaller. Inhibition of the UV-induced activation of Erbb2 also resulted in milder epidermal hyperplasia, S-phase accumulation, and decreased levels of the cell cycle regulator Cdc25a, suggesting altered cell cycle regulation on inhibition of Erbb2. Further investigation using inhibition or genetic deletion of Erbb2 in vitro revealed reduced Cdc25a levels and increased S-phase arrest in UV-irradiated cells lacking Erbb2 activity. Ectopic expression of Cdc25a prevented UV-induced S-phase arrest in keratinocytes lacking Erbb2 activity, demonstrating that maintenance of Cdc25a by Erbb2 suppresses cell cycle arrest. Examination of checkpoint pathway activation upstream of Cdc25a revealed Erbb2 activation did not alter Ataxia Telangiectasia and Rad3-related/Ataxia Telangiectasia Mutated activity but increased inhibitory phosphorylation of Chk1-Ser(280). Since Akt phosphorylates Chk1-Ser(280), the effect of Erbb2 on phosphatidyl inositol-3-kinase (PI3K)/Akt signaling during UV-induced cell cycle arrest was determined. Erbb2 ablation reduced the UV-induced activation of PI3K while inhibition of PI3K/Akt increased UV-induced S-phase arrest. Thus, UV-induced Erbb2 activation increases skin tumorigenesis through inhibitory phosphorylation of Chk1, Cdc25a maintenance, and suppression of S-phase arrest via a PI3K/Akt-dependent mechanism.

TLR5 participates in the TLR4 receptor complex and promotes MyD88-dependent signaling in environmental lung injury.

Lung disease causes significant morbidity and mortality, and is exacerbated by environmental injury, for example through lipopolysaccharide (LPS) or ozone (O3). Toll-like receptors (TLRs) orchestrate immune responses to injury by recognizing pathogen- or danger-associated molecular patterns. TLR4, the prototypic receptor for LPS, also mediates inflammation after O3, triggered by endogenous hyaluronan. Regulation of TLR4 signaling is incompletely understood. TLR5, the flagellin receptor, is expressed in alveolar macrophages, and regulates immune responses to environmental injury. Using in vivo animal models of TLR4-mediated inflammations (LPS, O3, hyaluronan), we show that TLR5 impacts the in vivo response to LPS, hyaluronan and O3. We demonstrate that immune cells of human carriers of a dominant negative TLR5 allele have decreased inflammatory response to O3 exposure ex vivo and LPS exposure in vitro. Using primary murine macrophages, we find that TLR5 physically associates with TLR4 and biases TLR4 signaling towards the MyD88 pathway. Our results suggest an updated paradigm for TLR4/TLR5 signaling.

Immune cells in the lung help guard against infections. On the surface of these cells are proteins called TLR receptors that recognize dangerous molecules or DNA from disease-causing microbes such as bacteria. When the immune cells detect these invaders, the TLR receptors spring into action and trigger an inflammatory response to destroy the microbes. This inflammation usually helps the lung clear infections. But it can also be harmful and damage the lung, for example when inflammation is caused by non-infectious substances such as pollutants in the atmosphere. There are several TLR receptors that each recognize a specific molecule. In 2010, researchers showed that the receptor TLR4 is responsible for causing inflammation in the lung after exposure to pollution. Another receptor called TLR5 also helps activate the immune response in the lung. But it was unclear whether this receptor also plays a role in pollution-linked lung damage. Now, Hussain, Johnson, Sciurba et al. ­ including one of the researchers involved in the 2010 study ­ have investigated the role of TLR5 in immune cells from the lungs of humans and mice. The experiments showed that TLR5 works together with TLR4 and helps trigger an inflammatory response to both pollutants and bacteria. Hussain et al. found that people lacking a working TLR5 receptor (which make up 3­10% of the population) are less likely to experience lung inflammation when exposed to pollution or bacterial proteins that activate TLR4. These findings suggest that people without TLR5 may be protected from pollution-induced lung injury. Further research into the role of TLR5 could help develop genetic tests for identifying people who are more sensitive to damage from pollution. This information could then be used to determine the likelihood of a patient experiencing certain lung diseases.

The prostaglandin receptor EP2 activates multiple signaling pathways and beta-arrestin1 complex formation during mouse skin papilloma development.

Prostaglandin E(2) (PGE(2)) is elevated in many tumor types, but PGE(2)'s contributions to tumor growth are largely unknown. To investigate PGE(2)'s roles, the contributions of one of its receptors, EP2, were studied using the mouse skin initiation/promotion model. Initial studies indicated that protein kinase A (PKA), epidermal growth factor receptor (EGFR) and several effectors-cyclic adenosine 3',5'-monophosphate response element-binding protein (CREB), H-Ras, Src, protein kinase B (AKT) and extracellular signal-regulated kinase (ERK)1/2-were activated in 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted papillomas and that PKA and EGFR inhibition (H89 and AG1478, respectively) decreased papilloma formation. EP2's contributions to the activation of these pathways and papilloma development were determined by inhibiting endogenous TPA-induced PGE(2) production with indomethacin (Indo) and concomitantly treating with the EP2 agonist, CAY10399 (CAY). CAY treatment restored papilloma formation in TPA/Indo-treated mice and increased cyclic adenosine 3',5'-monophosphate and PKA activation as measured by p-CREB formation. CAY treatment also increased EGFR and Src activation and their inhibition by AG1478 and PP2 indicated that Src was upstream of EGFR. CAY also increased H-Ras, ERK1/2 and AKT activation, and AG1478 decreased their activation indicating EGFR being upstream. Supporting EP2's contribution, EP2-/- mice exhibited 65% fewer papillomas and reduced Src, EGFR, H-Ras, AKT and ERK1/2 activation. G protein-coupled receptor (GPCR) activation of EGFR has been reported to involve Src's activation via a GPCR-beta-arrestin-Src complex. Indeed, immunoprecipitation of beta-arrestin1 or p-Src indicated the presence of an EP2-beta-arrestin1-p-Src complex in papillomas. The data indicated that EP2 contributed to tumor formation via activation of PKA and EGFR and that EP2 formed a complex with beta-arrestin1 and Src that contributed to signaling and/or EP2 desensitization.

Serum inter-alpha-trypsin inhibitor and matrix hyaluronan promote angiogenesis in fibrotic lung injury.

RATIONALE: The etiology and pathogenesis of angiogenesis in idiopathic pulmonary fibrosis (IPF) is poorly understood. Inter-alpha-trypsin inhibitor (IaI) is a serum protein that can bind to hyaluronan (HA) and may contribute to the angiogenic response to tissue injury. OBJECTIVES: To determine whether IaI promotes HA-mediated angiogenesis in tissue injury. METHODS: An examination was undertaken of angiogenesis in IaI-sufficient and -deficient mice in the bleomycin model of pulmonary fibrosis and in angiogenesis assays in vivo and in vitro. IaI and HA in patients with IPF were examined. MEASUREMENTS AND MAIN RESULTS: IaI significantly enhances the angiogenic response to short-fragment HA in vivo and in vitro. lal deficiency Ieads to decreased angiogenesis in the matrigel model, and decreases lung angiogenesis after bleomycin exposure in mice. IaI is found in fibroblastic foci in IPF, where it colocalizes with HA. The colocalization is particularly strong in vascular areas around fibroblastic foci. Serum levels of IaI and HA are significantly elevated in patients with IPF compared with control subjects. High serum IaI and HA levels are associated with decreased lung diffusing capacity, but not FVC. CONCLUSIONS: Our findings indicate that serum IaI interacts with HA, and promotes angiogenesis in lung injury. IaI appears to contribute to the vascular response to lung injury and may lead to aberrant angiogenesis. Clinical trial registered with www.clinicaltrials.gov (NCT00016627).

CD34 expression by hair follicle stem cells is required for skin tumor development in mice.

The cell surface marker CD34 marks mouse hair follicle bulge cells, which have attributes of stem cells, including quiescence and multipotency. Using a CD34 knockout (KO) mouse, we tested the hypothesis that CD34 may participate in tumor development in mice because hair follicle stem cells are thought to be a major target of carcinogens in the two-stage model of mouse skin carcinogenesis. Following initiation with 200 nmol 7,12-dimethylbenz(a)anthracene (DMBA), mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 20 weeks. Under these conditions, CD34KO mice failed to develop papillomas. Increasing the initiating dose of DMBA to 400 nmol resulted in tumor development in the CD34KO mice, albeit with an increased latency and lower tumor yield compared with the wild-type (WT) strain. DNA adduct analysis of keratinocytes from DMBA-initiated CD34KO mice revealed that DMBA was metabolically activated into carcinogenic diol epoxides at both 200 and 400 nmol. Chronic exposure to TPA revealed that CD34KO skin developed and sustained epidermal hyperplasia. However, CD34KO hair follicles typically remained in telogen rather than transitioning into anagen growth, confirmed by retention of bromodeoxyuridine-labeled bulge stem cells within the hair follicle. Unique localization of the hair follicle progenitor cell marker MTS24 was found in interfollicular basal cells in TPA-treated WT mice, whereas staining remained restricted to the hair follicles of CD34KO mice, suggesting that progenitor cells migrate into epidermis differently between strains. These data show that CD34 is required for TPA-induced hair follicle stem cell activation and tumor formation in mice.

Bone marrow-derived epithelial cells and hair follicle stem cells contribute to development of chronic cutaneous neoplasms.

We used allogeneic bone marrow transplantation (BMT) and a mouse multistage cutaneous carcinogenesis model to probe recruitment of bone marrow-derived epithelial cells (BMDECs) in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with 12-O-tetradecanolyphorbol-13-acetate (TPA). BMDECs clustered in the lesional epithelium, expressed cytokeratins, proliferated, and stratified. We detected cytokeratin induction in plastic-adherent bone marrow cells (BMCs) cultured in the presence of filter-separated keratinocytes (KCs) and bone morphogenetic protein 5 (BMP5). Lineage-depleted BMCs migrated towards High Mobility Group Box 1 (HMGB1) protein and epidermal KCs in ex vivo invasion assays. Naive female mice receiving BMTs from DMBA-treated donors developed benign and malignant lesions after TPA promotion alone. We conclude that BMDECs contribute to the development of papillomas and dysplasia, demonstrating a systemic contribution to these lesions. Furthermore, carcinogen-exposed BMCs can initiate benign and malignant lesions upon tumor promotion. Ultimately, these findings may suggest targets for treatment of non-melanoma skin cancers.