RESUMO
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes that complete the first step of protein translation: ligation of amino acids to cognate tRNAs. Genes encoding ARSs have been implicated in myriad dominant and recessive phenotypes, the latter often affecting multiple tissues but with frequent involvement of the central and peripheral nervous systems, liver, and lungs. Threonyl-tRNA synthetase (TARS1) encodes the enzyme that ligates threonine to tRNATHR in the cytoplasm. To date, TARS1 variants have been implicated in a recessive brittle hair phenotype. To better understand TARS1-related recessive phenotypes, we engineered three TARS1 missense variants at conserved residues and studied these variants in Saccharomyces cerevisiae and Caenorhabditis elegans models. This revealed two loss-of-function variants, including one hypomorphic allele (R433H). We next used R433H to study the effects of partial loss of TARS1 function in a compound heterozygous mouse model (R432H/null). This model presents with phenotypes reminiscent of patients with TARS1 variants and with distinct lung and skin defects. This study expands the potential clinical heterogeneity of TARS1-related recessive disease, which should guide future clinical and genetic evaluations of patient populations.
Assuntos
Caenorhabditis elegans , Saccharomyces cerevisiae , Treonina-tRNA Ligase , Animais , Camundongos , Caenorhabditis elegans/genética , Saccharomyces cerevisiae/genética , Treonina-tRNA Ligase/genética , Treonina-tRNA Ligase/metabolismo , Humanos , Fenótipo , Mutação com Perda de Função , Modelos Animais de Doenças , Mutação de Sentido IncorretoRESUMO
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes that complete the first step of protein translation: ligation of amino acids to cognate tRNAs. Genes encoding ARSs have been implicated in myriad dominant and recessive phenotypes, the latter often affecting multiple tissues but with frequent involvement of the central and peripheral nervous system, liver, and lungs. Threonyl-tRNA synthetase (TARS1) encodes the enzyme that ligates threonine to tRNATHR in the cytoplasm. To date, TARS1 variants have been implicated in a recessive brittle hair phenotype. To better understand TARS1-related recessive phenotypes, we engineered three TARS1 missense mutations predicted to cause a loss-of-function effect and studied these variants in yeast and worm models. This revealed two loss-of-function mutations, including one hypomorphic allele (R433H). We next used R433H to study the effects of partial loss of TARS1 function in a compound heterozygous mouse model (R433H/null). This model presents with phenotypes reminiscent of patients with TARS1 variants and with distinct lung and skin defects. This study expands the potential clinical heterogeneity of TARS1-related recessive disease, which should guide future clinical and genetic evaluations of patient populations.
RESUMO
Sebaceous glands (SGs) release oils that protect our skin, but how these glands respond to injury has not been previously examined. Here, we report that SGs are largely self-renewed by dedicated stem cell pools during homeostasis. Using targeted single-cell RNA sequencing, we uncovered both direct and indirect paths by which resident SG progenitors ordinarily differentiate into sebocytes, including transit through a Krt5+PPARγ+ transitional basal cell state. Upon skin injury, however, SG progenitors depart their niche, reepithelialize the wound, and are replaced by hair-follicle-derived stem cells. Furthermore, following targeted genetic ablation of >99% of SGs from dorsal skin, these glands unexpectedly regenerate within weeks. This regenerative process is mediated by alternative stem cells originating from the hair follicle bulge, is dependent upon FGFR2 signaling, and can be accelerated by inducing hair growth. Altogether, our studies demonstrate that stem cell plasticity promotes SG durability following injury.
Assuntos
Glândulas Sebáceas , Pele , Diferenciação Celular , Folículo Piloso , Células EpiteliaisRESUMO
Sebaceous glands (SGs) release oils that protect our skin, but how these glands respond to injury has not been previously examined. Here, we report that SGs are largely self-renewed by dedicated stem cell pools during homeostasis. Using targeted single cell RNA-sequencing, we uncovered both direct and indirect paths by which these resident SG progenitors ordinarily differentiate into sebocytes, including transit through a PPARγ+Krt5+ transitional cell state. Upon skin injury, however, SG progenitors depart their niche, reepithelialize the wound, and are replaced by hair follicle-derived stem cells. Furthermore, following targeted genetic ablation of >99% of SGs from dorsal skin, these glands unexpectedly regenerate within weeks. This regenerative process is mediated by alternative stem cells originating from the hair follicle bulge, is dependent upon FGFR signaling, and can be accelerated by inducing hair growth. Altogether, our studies demonstrate that stem cell plasticity promotes SG durability following injury.
RESUMO
Basal cell carcinomas (BCCs) frequently possess immense mutational burdens; however, the functional significance of most of these mutations remains unclear. Here, we report that loss of Ptch1, the most common mutation that activates upstream Hedgehog (Hh) signaling, initiates the formation of nascent BCC-like tumors that eventually enter into a dormant state. However, rare tumors that overcome dormancy acquire the ability to hyperactivate downstream Hh signaling through a variety of mechanisms, including amplification of Gli1/2 and upregulation of Mycn. Furthermore, we demonstrate that MYCN overexpression promotes the progression of tumors induced by loss of Ptch1. These findings suggest that canonical mutations that activate upstream Hh signaling are necessary, but not sufficient, for BCC to fully progress. Rather, tumors likely acquire secondary mutations that further hyperactivate downstream Hh signaling in order to escape dormancy and enter a trajectory of uncontrolled expansion.
Assuntos
Carcinoma Basocelular , Neoplasias Cutâneas , Carcinoma Basocelular/genética , Carcinoma Basocelular/patologia , Proteínas Hedgehog/genética , Humanos , Mutação/genética , Proteína Proto-Oncogênica N-Myc/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer that frequently carries an integrated Merkel cell polyomavirus (MCPyV) genome and expresses viral transforming antigens (TAgs). MCC tumor cells also express signature genes detected in skin-resident, postmitotic Merkel cells, including atonal bHLH transcription factor 1 (ATOH1), which is required for Merkel cell development from epidermal progenitors. We now report the use of in vivo cellular reprogramming, using ATOH1, to drive MCC development from murine epidermis. We generated mice that conditionally expressed MCPyV TAgs and ATOH1 in epidermal cells, yielding microscopic collections of proliferating MCC-like cells arising from hair follicles. Immunostaining of these nascent tumors revealed p53 accumulation and apoptosis, and targeted deletion of transformation related protein 53 (Trp53) led to development of gross skin tumors with classic MCC histology and marker expression. Global transcriptome analysis confirmed the close similarity of mouse and human MCCs, and hierarchical clustering showed conserved upregulation of signature genes. Our data establish that expression of MCPyV TAgs in ATOH1-reprogrammed epidermal cells and their neuroendocrine progeny initiates hair follicle-derived MCC tumorigenesis in adult mice. Moreover, progression to full-blown MCC in this model requires loss of p53, mimicking the functional inhibition of p53 reported in human MCPyV-positive MCCs.
Assuntos
Carcinoma de Célula de Merkel , Poliomavírus das Células de Merkel , Infecções por Polyomavirus , Neoplasias Cutâneas , Infecções Tumorais por Vírus , Animais , Antígenos Virais , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/metabolismo , Carcinoma de Célula de Merkel/patologia , Reprogramação Celular , Poliomavírus das Células de Merkel/genética , Camundongos , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/patologia , Neoplasias Cutâneas/patologia , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/patologiaRESUMO
HDAC inhibitors show therapeutic promise for skin malignancies; however, the roles of specific HDACs in adult epidermal homeostasis and in disease are poorly understood. We find that homozygous epidermal codeletion of Hdac1 and Hdac2 in adult mouse epidermis causes reduced basal cell proliferation, apoptosis, inappropriate differentiation, and eventual loss of Hdac1/2-null keratinocytes. Hdac1/2-deficient epidermis displays elevated acetylated p53 and increased expression of the senescence gene p16. Loss of p53 partially restores basal proliferation, whereas p16 deletion promotes long-term survival of Hdac1/2-null keratinocytes. In activated GLI2-driven pre-basal cell carcinoma, Hdac1/2 deletion dramatically reduces proliferation and increases apoptosis, and knockout of either p53 or p16 partially rescues both proliferation and basal cell viability. Topical application of the HDAC inhibitor romidepsin to the normal epidermis or to GLI2ΔN-driven lesions produces similar defects to those caused by genetic Hdac1/2 deletion, and these are partially rescued by loss of p16. These data reveal essential roles for HDAC1/2 in maintaining proliferation and survival of adult epidermal and basal cell carcinoma progenitors and suggest that the efficacy of therapeutic HDAC1/2 inhibition will depend in part on the mutational status of p53 and p16.
Assuntos
Carcinoma Basocelular/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Epiderme/fisiologia , Queratinócitos/fisiologia , Neoplasias Cutâneas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/uso terapêutico , Apoptose , Carcinogênese , Carcinoma Basocelular/tratamento farmacológico , Carcinoma Basocelular/genética , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/genética , Depsipeptídeos/farmacologia , Depsipeptídeos/uso terapêutico , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/genética , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Lesões Pré-Cancerosas , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine carcinoma without a known dysplastic precursor. In some cases, MCC is associated with SCCIS in the overlying epidermis; however, the MCC and SCCIS populations display strikingly different morphologies, and thus far a relationship between these components has not been demonstrated. To better understand the relationship between these distinct tumor cell populations, we evaluated 7 pairs of MCC-SCCIS for overlapping genomic alterations by cancer profiling panel. A subset was further characterized by transcriptional profiling and immunohistochemistry. In 6 of 7 MCC-SCCIS pairs there was highly significant mutational overlap including shared TP53 and/or RB1 mutations. In some cases, oncogenic events previously implicated in MCC (MYCL gain, MDM4 gain, HRAS mutation) were detected in both components. Although FBXW7 mutations were enriched in MCC, no gene mutation was unique to the MCC component across all cases. Transcriptome analysis identified 2736 differentially expressed genes between MCC and SCCIS. Genes upregulated in the MCC component included Polycomb repressive complex targets; downregulated transcripts included epidermal markers, and immune genes such as HLA-A. Immunohistochemical studies revealed increased expression of SOX2 in the MCC component, with diminished H3K27Me3, Rb, and HLA-A expression. In summary, MCC-SCCIS pairs demonstrate clonal relatedness. The shift to neuroendocrine phenotype is associated with loss of Rb protein expression, decrease in global H3K27Me3, and increased expression of Merkel cell genes such as SOX2. Our findings suggest an epidermal origin of MCC in this setting, and to our knowledge provide the first molecular evidence that intraepithelial squamous dysplasia may represent a direct precursor for small cell carcinoma.
Assuntos
Carcinoma de Célula de Merkel , Carcinoma de Células Escamosas , Neoplasias Cutâneas , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/patologia , Carcinoma de Células Escamosas/genética , Proteínas de Ciclo Celular , Genômica , Humanos , Imuno-Histoquímica , Proteínas Proto-Oncogênicas , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologiaRESUMO
Obesity is a worldwide epidemic that predisposes individuals to many age-associated diseases, but its exact effects on organ dysfunction are largely unknown1. Hair follicles-mini-epithelial organs that grow hair-are miniaturized by ageing to cause hair loss through the depletion of hair follicle stem cells (HFSCs)2. Here we report that obesity-induced stress, such as that induced by a high-fat diet (HFD), targets HFSCs to accelerate hair thinning. Chronological gene expression analysis revealed that HFD feeding for four consecutive days in young mice directed activated HFSCs towards epidermal keratinization by generating excess reactive oxygen species, but did not reduce the pool of HFSCs. Integrative analysis using stem cell fate tracing, epigenetics and reverse genetics showed that further feeding with an HFD subsequently induced lipid droplets and NF-κB activation within HFSCs via autocrine and/or paracrine IL-1R signalling. These integrated factors converge on the marked inhibition of Sonic hedgehog (SHH) signal transduction in HFSCs, thereby further depleting lipid-laden HFSCs through their aberrant differentiation and inducing hair follicle miniaturization and eventual hair loss. Conversely, transgenic or pharmacological activation of SHH rescued HFD-induced hair loss. These data collectively demonstrate that stem cell inflammatory signals induced by obesity robustly represses organ regeneration signals to accelerate the miniaturization of mini-organs, and suggests the importance of daily prevention of organ dysfunction.
Assuntos
Alopecia/patologia , Alopecia/fisiopatologia , Folículo Piloso/patologia , Obesidade/fisiopatologia , Células-Tronco/patologia , Animais , Comunicação Autócrina , Contagem de Células , Diferenciação Celular , Linhagem da Célula , Senescência Celular , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Proteínas Hedgehog/metabolismo , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/patologia , Estresse Oxidativo , Comunicação Parácrina , Receptores de Interleucina-1/metabolismoRESUMO
Uncontrolled activation of the Hedgehog (Hh) signaling pathway, operating through GLI transcription factors, plays a central role in the pathogenesis of cutaneous basal cell carcinoma and contributes to the development of several malignancies arising in extracutaneous sites. We now report that K5-tTA;tetO-Gli2 bitransgenic mice develop distinctive epithelial tumors within their jaws. These tumors consist of large masses of highly proliferative, monomorphous, basaloid cells with scattered foci of keratinization and central necrosis, mimicking human basaloid squamous cell carcinoma (BSCC), an aggressive upper aerodigestive tract tumor. Like human BSCC, these tumors express epidermal basal keratins and differentiation-specific keratins within squamous foci. Mouse BSCCs express high levels of Gli2 and Hh target genes, including Gli1 and Ptch1, which we show are also upregulated in a subset of human BSCCs. Mouse BSCCs appear to arise from distinct epithelial sites, including the gingival junctional epithelium and epithelial rests of Malassez, a proposed stem cell compartment. Although Gli2 transgene expression is restricted to epithelial cells, we also detect striking alterations in bone adjacent to BSCCs, with activated osteoblasts, osteoclasts and osteal macrophages, indicative of active bone remodeling. Gli2 transgene inactivation resulted in rapid BSCC regression and reversal of the bone remodeling phenotype. This first-reported mouse model of BSCC supports the concept that uncontrolled Hh signaling plays a central role in the pathogenesis of a subset of human BSCCs, points to Hh/GLI2 signaling as a potential therapeutic target and provides a powerful new tool for probing the mechanistic underpinnings of tumor-associated bone remodeling.
Assuntos
Remodelação Óssea , Carcinoma de Células Escamosas/patologia , Proteínas Hedgehog/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Cutâneas/patologia , Proteína Gli2 com Dedos de Zinco/metabolismo , Animais , Carcinoma de Células Escamosas/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Cutâneas/metabolismoRESUMO
Oil-secreting sebaceous glands (SGs) are critical for proper skin function; however, it remains unclear how different factors act together to modulate SG stem cells. Here, we provide functional evidence that each SG lobe is serviced by its own dedicated stem cell population. Upon ablating Notch signaling in different skin subcompartments, we find that this pathway exerts dual counteracting effects on SGs. Suppressing Notch in SG progenitors traps them in a hybrid state where stem and differentiation features become intermingled. In contrast, ablating Notch outside of the SG stem cell compartment indirectly drives SG expansion. Finally, we report that a K14:K5âK14:K79 keratin shift occurs during SG differentiation. Deleting K79 destabilizes K14 in sebocytes, and attenuates SGs and eyelid meibomian glands, leading to corneal ulceration. Altogether, our findings demonstrate that SGs integrate diverse signals from different niches and suggest that mutations incurred within one stem cell compartment can indirectly influence another.
Assuntos
Glândulas Sebáceas/citologia , Pele/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Animais , Diferenciação Celular , Feminino , Proteínas Hedgehog/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Queratinas/metabolismo , Masculino , Glândulas Tarsais/metabolismo , Camundongos Knockout , Mutação/genética , Receptores Notch/genéticaRESUMO
Striking taste disturbances are reported in cancer patients treated with Hedgehog (HH)-pathway inhibitor drugs, including sonidegib (LDE225), which block the HH pathway effector Smoothened (SMO). We tested the potential for molecular, cellular, and functional recovery in mice from the severe disruption of taste-organ biology and taste sensation that follows HH/SMO signaling inhibition. Sonidegib treatment led to rapid loss of taste buds (TB) in both fungiform and circumvallate papillae, including disruption of TB progenitor-cell proliferation and differentiation. Effects were selective, sparing nontaste papillae. To confirm that taste-organ effects of sonidegib treatment result from HH/SMO signaling inhibition, we studied mice with conditional global or epithelium-specific Smo deletions and observed similar effects. During sonidegib treatment, chorda tympani nerve responses to lingual chemical stimulation were maintained at 10 d but were eliminated after 16 d, associated with nearly complete TB loss. Notably, responses to tactile or cold stimulus modalities were retained. Further, innervation, which was maintained in the papilla core throughout treatment, was not sufficient to sustain TB during HH/SMO inhibition. Importantly, treatment cessation led to rapid and complete restoration of taste responses within 14 d associated with morphologic recovery in about 55% of TB. However, although taste nerve responses were sustained, TB were not restored in all fungiform papillae even with prolonged recovery for several months. This study establishes a physiologic, selective requirement for HH/SMO signaling in taste homeostasis that includes potential for sensory restoration and can explain the temporal recovery after taste dysgeusia in patients treated with HH/SMO inhibitors.
Assuntos
Antineoplásicos/efeitos adversos , Compostos de Bifenilo/efeitos adversos , Disgeusia/fisiopatologia , Piridinas/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Paladar/efeitos dos fármacos , Língua/fisiopatologia , Animais , Carcinoma Basocelular/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Nervo da Corda do Tímpano/efeitos dos fármacos , Nervo da Corda do Tímpano/fisiopatologia , Modelos Animais de Doenças , Disgeusia/induzido quimicamente , Disgeusia/patologia , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Recuperação de Função Fisiológica , Neoplasias Cutâneas/tratamento farmacológico , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Células-Tronco/efeitos dos fármacos , Paladar/fisiologia , Papilas Gustativas/citologia , Papilas Gustativas/efeitos dos fármacos , Papilas Gustativas/patologia , Papilas Gustativas/fisiopatologia , Língua/efeitos dos fármacos , Língua/inervaçãoRESUMO
For homeostasis, lingual taste papilla organs require regulation of epithelial cell survival and renewal, with sustained innervation and stromal interactions. To investigate a role for Hedgehog/GLI signaling in adult taste organs we used a panel of conditional mouse models to manipulate GLI activity within epithelial cells of the fungiform and circumvallate papillae. Hedgehog signaling suppression rapidly led to taste bud loss, papilla disruption, and decreased proliferation in domains of papilla epithelium that contribute to taste cells. Hedgehog responding cells were eliminated from the epithelium but retained in the papilla stromal core. Despite papilla disruption and loss of taste buds that are a major source of Hedgehog ligand, innervation to taste papillae was maintained, and not misdirected, even after prolonged GLI blockade. Further, vimentin-positive fibroblasts remained in the papilla core. However, retained innervation and stromal cells were not sufficient to maintain taste bud cells in the context of compromised epithelial Hedgehog signaling. Importantly taste organ disruption after GLI blockade was reversible in papillae that retained some taste bud cell remnants where reactivation of Hedgehog signaling led to regeneration of papilla epithelium and taste buds. Therefore, taste bud progenitors were either retained during epithelial GLI blockade or readily repopulated during recovery, and were poised to regenerate taste buds once Hedgehog signaling was restored, with innervation and papilla connective tissue elements in place. Our data argue that Hedgehog signaling is essential for adult tongue tissue maintenance and that taste papilla epithelial cells represent the key targets for physiologic Hedgehog-dependent regulation of taste organ homeostasis. Because disruption of GLI transcriptional activity in taste papilla epithelium is sufficient to drive taste organ loss, similar to pharmacologic Hedgehog pathway inhibition, the findings suggest that taste alterations in cancer patients using systemic Hedgehog pathway inhibitors result principally from interruption of signaling activity in taste papillae.
Assuntos
Proteínas Hedgehog/genética , Papilas Gustativas/metabolismo , Paladar/genética , Língua/metabolismo , Animais , Células Epiteliais/metabolismo , Epitélio/metabolismo , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Camundongos , Fibras Nervosas/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Papilas Gustativas/crescimento & desenvolvimento , Percepção Gustatória/genéticaRESUMO
Gastric adenocarcinoma is the third most common cause of cancer-related death worldwide. Here we report a novel, highly-penetrant mouse model of invasive gastric cancer arising from deregulated Hedgehog/Gli2 signaling targeted to Lgr5-expressing stem cells in adult stomach. Tumor development progressed rapidly: three weeks after inducing the Hh pathway oncogene GLI2A, 65% of mice harbored in situ gastric cancer, and an additional 23% of mice had locally invasive tumors. Advanced mouse gastric tumors had multiple features in common with human gastric adenocarcinomas, including characteristic histological changes, expression of RNA and protein markers, and the presence of major inflammatory and stromal cell populations. A subset of tumor cells underwent epithelial-mesenchymal transition, likely mediated by focal activation of canonical Wnt signaling and Snail1 induction. Strikingly, mTOR pathway activation, based on pS6 expression, was robustly activated in mouse gastric adenocarcinomas from the earliest stages of tumor development, and treatment with rapamycin impaired tumor growth. GLI2A-expressing epithelial cells were detected transiently in intestine, which also contains Lgr5+ stem cells, but they did not give rise to epithelial tumors in this organ. These findings establish that deregulated activation of Hedgehog/Gli2 signaling in Lgr5-expressing stem cells is sufficient to drive gastric adenocarcinoma development in mice, identify a critical requirement for mTOR signaling in the pathogenesis of these tumors, and underscore the importance of tissue context in defining stem cell responsiveness to oncogenic stimuli.
Assuntos
Adenocarcinoma/patologia , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Neoplásicas/patologia , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Gástricas/patologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Nus , Proteínas Quinases S6 Ribossômicas/metabolismo , Sirolimo/uso terapêutico , Fatores de Transcrição da Família Snail/metabolismo , Via de Sinalização Wnt , Proteína Gli2 com Dedos de Zinco , beta Catenina/metabolismoRESUMO
To explore the role of amphiregulin in inflammatory epidermal hyperplasia, we overexpressed human AREG (hAREG) in FVB/N mice using a bovine K5 promoter. A construct containing AREG coding sequences flanked by 5' and 3' untranslated region sequences (AREG-UTR) led to a >10-fold increase in hAREG expression compared to an otherwise-identical construct containing only the coding region (AREG-CDR). AREG-UTR mice developed tousled, greasy fur as well as elongated nails and thickened, erythematous tail skin. No such phenotype was evident in AREG-CDR mice. Histologically, AREG-UTR mice presented with marked epidermal hyperplasia of tail skin (2.1-fold increase in epidermal thickness with a 9.5-fold increase in Ki-67(+) cells) accompanied by significantly increased CD4+ T-cell infiltration. Dorsal skin of AREG-UTR mice manifested lesser but still significant increases in epidermal thickness and keratinocyte hyperplasia. AREG-UTR mice also developed marked and significant sebaceous gland enlargement, with corresponding increases in Ki-67(+) cells. To determine the response of AREG-UTR animals to a pro-inflammatory skin challenge, topical imiquimod (IMQ) or vehicle cream was applied to dorsal and tail skin. IMQ increased dorsal skin thickness similarly in both AREG-UTR and wild type mice (1.7- and 2.2-fold vs vehicle, P < 0.001 each), but had no such effect on tail skin. These results confirm that keratinocyte expression of hAREG elicits inflammatory epidermal hyperplasia, and are consistent with prior reports of tail epidermal hyperplasia and increased sebaceous gland size in mice expressing human epigen.
Assuntos
Anfirregulina/genética , Anfirregulina/metabolismo , Pele/metabolismo , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Bovinos , Epiderme/patologia , Receptores ErbB/metabolismo , Homeostase , Humanos , Hiperplasia/metabolismo , Inflamação , Ligantes , Camundongos , Camundongos Transgênicos , Fenótipo , Regiões Promotoras Genéticas , Glândulas Sebáceas/metabolismoRESUMO
The adult fungiform taste papilla is a complex of specialized cell types residing in the stratified squamous tongue epithelium. This unique sensory organ includes taste buds, papilla epithelium and lateral walls that extend into underlying connective tissue to surround a core of lamina propria cells. Fungiform papillae must contain long-lived, sustaining or stem cells and short-lived, maintaining or transit amplifying cells that support the papilla and specialized taste buds. Shh signaling has established roles in supporting fungiform induction, development and patterning. However, for a full understanding of how Shh transduced signals act in tongue, papilla and taste bud formation and maintenance, it is necessary to know where and when the Shh ligand and pathway components are positioned. We used immunostaining, in situ hybridization and mouse reporter strains for Shh, Ptch1, Gli1 and Gli2-expression and proliferation markers to identify cells that participate in hedgehog signaling. Whereas there is a progressive restriction in location of Shh ligand-expressing cells, from placode and apical papilla cells to taste bud cells only, a surrounding population of Ptch1 and Gli1 responding cells is maintained in signaling centers throughout papilla and taste bud development and differentiation. The Shh signaling targets are in regions of active cell proliferation. Using genetic-inducible lineage tracing for Gli1-expression, we found that Shh-responding cells contribute not only to maintenance of filiform and fungiform papillae, but also to taste buds. A requirement for normal Shh signaling in fungiform papilla, taste bud and filiform papilla maintenance was shown by Gli2 constitutive activation. We identified proliferation niches where Shh signaling is active and suggest that epithelial and mesenchymal compartments harbor potential stem and/or progenitor cell zones. In all, we report a set of hedgehog signaling centers that regulate development and maintenance of taste organs, the fungiform papilla and taste bud, and surrounding lingual cells. Shh signaling has roles in forming and maintaining fungiform papillae and taste buds, most likely via stage-specific autocrine and/or paracrine mechanisms, and by engaging epithelial/mesenchymal interactions.
Assuntos
Epitélio/embriologia , Epitélio/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Papilas Gustativas/embriologia , Papilas Gustativas/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Compartimento Celular , Linhagem da Célula , Proliferação de Células , Microambiente Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Fatores de Transcrição Kruppel-Like/metabolismo , Ligantes , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/metabolismo , Papilas Gustativas/citologia , Papilas Gustativas/ultraestrutura , Fatores de Tempo , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de ZincoRESUMO
Gastric adenocarcinoma is one of the leading causes of cancer mortality worldwide. It arises through a stepwise process that includes prominent inflammation with expression of interferon-γ (IFN-γ) and multiple other pro-inflammatory cytokines. We engineered mice expressing IFN-γ under the control of the stomach-specific H(+)/K(+) ATPase ß promoter to test the potential role of this cytokine in gastric tumorigenesis. Stomachs of H/K-IFN-γ transgenic mice exhibited inflammation, expansion of myofibroblasts, loss of parietal and chief cells, spasmolytic polypeptide expressing metaplasia, and dysplasia. Proliferation was elevated in undifferentiated and metaplastic epithelial cells in H/K-IFN-γ transgenic mice, and there was increased apoptosis. H/K-IFN-γ mice had elevated levels of mRNA for IFN-γ target genes and the pro-inflammatory cytokines IL-6, IL-1ß, and tumor necrosis factor-α. Intracellular mediators of IFN-γ and IL-6 signaling, pSTAT1 and pSTAT3, respectively, were detected in multiple cell types within stomach. H/K-IFN-γ mice developed dysplasia as early as 3 months of age, and 4 of 39 mice over 1 year of age developed antral polyps or tumors, including one adenoma and one adenocarcinoma, which expressed high levels of nuclear ß-catenin. Our data identified IFN-γ as a pivotal secreted factor that orchestrates complex changes in inflammatory, epithelial, and mesenchymal cell populations to drive pre-neoplastic progression in stomach; however, additional alterations appear to be required for malignant conversion.
Assuntos
Mucosa Gástrica/metabolismo , Inflamação/patologia , Interferon gama/genética , Estômago/patologia , Animais , Apoptose/genética , Atrofia , Linhagem da Célula/genética , Proliferação de Células , Progressão da Doença , Feminino , ATPase Trocadora de Hidrogênio-Potássio/genética , Proteínas Hedgehog/metabolismo , Inflamação/genética , Peptídeos e Proteínas de Sinalização Intercelular , Interferon gama/metabolismo , Masculino , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tamanho do Órgão , Células Parietais Gástricas/metabolismo , Células Parietais Gástricas/patologia , Peptídeos/metabolismo , Lesões Pré-Cancerosas/patologia , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Regulação para Cima/genéticaRESUMO
Uncontrolled Hedgehog (Hh) signaling leads to the development of basal cell carcinoma (BCC), the most common human cancer, but the cell of origin for BCC is unclear. While Hh pathway dysregulation is common to essentially all BCCs, there exist multiple histological subtypes, including superficial and nodular variants, raising the possibility that morphologically distinct BCCs may arise from different cellular compartments in skin. Here we have shown that induction of a major mediator of Hh signaling, GLI2 activator (GLI2ΔN), selectively in stem cells of resting hair follicles in mice, induced nodular BCC development from a small subset of cells in the lower bulge and secondary hair germ compartments. Tumorigenesis was markedly accelerated when GLI2ΔN was induced in growing hair follicles. In contrast, induction of GLI2ΔN in epidermis led to the formation of superficial BCCs. Expression of GLI2ΔN at reduced levels in mice yielded lesions resembling basaloid follicular hamartomas, which have previously been linked to low-level Hh signaling in both mice and humans. Our data show that the cell of origin, tissue context (quiescent versus growing hair follicles), and level of oncogenic signaling can determine the phenotype of Hh/Gli-driven skin tumors, with high-level signaling required for development of superficial BCC-like tumors from interfollicular epidermis and nodular BCC-like tumors from hair follicle stem cells.
Assuntos
Carcinoma Basocelular/metabolismo , Células Epiteliais/citologia , Folículo Piloso/metabolismo , Neoplasias Cutâneas/metabolismo , Células-Tronco/citologia , Alelos , Animais , Epiderme/metabolismo , Hamartoma/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Hiperplasia , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Transgênicos , Fenótipo , Estrutura Terciária de Proteína , Transdução de Sinais , Proteína Gli2 com Dedos de ZincoRESUMO
Bone morphogenetic proteins (BMPs) play pivotal roles in the regulation of skin development. To study the role of BMPs in skin tumorigenesis, BMP antagonist noggin was used to generate keratin 14-targeted transgenic mice. In contrast to wild-type mice, transgenic mice developed spontaneous hair follicle-derived tumors, which resemble human trichofolliculoma. Global gene expression profiles revealed that in contrast to anagen hair follicles of wild-type mice, tumors of transgenic mice showed stage-dependent increases in the expression of genes encoding the selected components of Wnt and Shh pathways. Specifically, expression of the Wnt ligands increased at the initiation stage of tumor formation, whereas expression of the Wnt antagonist and tumor suppressor Wnt inhibitory factor-1 decreased, as compared with fully developed tumors. In contrast, expression of the components of Shh pathway increased in fully developed tumors, as compared with the tumor placodes. Consistent with the expression data, pharmacological treatment of transgenic mice with Wnt and Shh antagonists resulted in the stage-dependent inhibition of tumor initiation, and progression, respectively. Furthermore, BMP signaling stimulated Wnt inhibitory factor-1 expression and promoter activity in cultured tumor cells and HaCaT keratinocytes, as well as inhibited Shh expression, as compared with the corresponding controls. Thus, tumor suppressor activity of the BMPs in skin epithelium depends on the local concentrations of noggin and is mediated at least in part via stage-dependent antagonizing of Wnt and Shh signaling pathways.
Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas de Transporte/genética , Neoplasias Cutâneas/metabolismo , Adulto , Idoso , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/biossíntese , Transformação Celular Neoplásica , Feminino , Folículo Piloso/metabolismo , Folículo Piloso/patologia , Proteínas Hedgehog/metabolismo , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Transdução de Sinais/genética , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologia , Proteínas Wnt/metabolismoRESUMO
Recent reports have suggested that the hedgehog (Hh) pathway is activated in lesional psoriatic skin, and that treatment with the Hh pathway antagonist cyclopamine may lead to rapid resolution of the disease. To assess Hh pathway activity in psoriasis, we isolated RNA from lesional and uninvolved skin of 58 psoriatic patients, and from 63 normal control subjects, and subjected these samples to global gene expression profiling on Affymetrix HU133 Plus 2.0 gene arrays. We were especially interested in Hh target genes (PTCH1 and GLI1), whose expression is elevated in response to Hh signaling. The microarray data demonstrated downregulation of PTCH1 expression in uninvolved and lesional skin (1.1-fold and 2-fold, respectively; P<0.0001). Additionally GLI1 mRNA was downregulated in lesional skin (1.7 fold; P<0.05). No significant changes were observed between lesional and uninvolved skin for the Hh ligands or Smoothened. Quantitative PCR confirmed these findings. In situ hybridization for GLI1 and PTCH1 was positive in basal cell carcinoma tumor cells, but was negligible in uninvolved or lesional psoriatic skin. The absence of elevated Hh target gene expression in lesional psoriatic skin indicates that the Hh pathway is not activated in this disease, raising questions regarding the proposed use of Hh antagonists as antipsoriatic agents.