RESUMO
Spermiogenesis is the final phase during sperm cell development in which round spermatids undergo dramatic morphological changes to generate spermatozoa. Here we report that the serine/threonine kinase Stk33 is essential for the differentiation of round spermatids into functional sperm cells and male fertility. Constitutive Stk33 deletion in mice results in severely malformed and immotile spermatozoa that are particularly characterized by disordered structural tail elements. Stk33 expression first appears in primary spermatocytes, and targeted deletion of Stk33 in these cells recapitulates the defects observed in constitutive knockout mice, confirming a germ cell-intrinsic function. Stk33 protein resides in the cytoplasm and partially co-localizes with the caudal end of the manchette, a transient structure that guides tail elongation, in elongating spermatids, and loss of Stk33 leads to the appearance of a tight, straight and elongated manchette. Together, these results identify Stk33 as an essential regulator of spermatid differentiation and male fertility.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Espermátides/enzimologia , Animais , Diferenciação Celular/fisiologia , Fertilidade/fisiologia , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Espermatócitos/citologia , Espermatócitos/enzimologia , Espermatogênese/fisiologia , Espermatozoides/enzimologia , Testículo/enzimologiaRESUMO
The angiopoietin (Angpt)-TIE signaling pathway controls vascular maturation and maintains the quiescent phenotype of resting vasculature. The contextual agonistic and antagonistic Tie2 ligand ANGPT2 is believed to be exclusively produced by endothelial cells, disrupting constitutive ANGPT1-TIE2 signaling to destabilize the microvasculature during pathologic disorders like inflammation and cancer. However, scattered reports have also portrayed tumor cells as a source of ANGPT2. Employing ISH-based detection of ANGPT2, we found strong tumor cell expression of ANGPT2 in a subset of patients with melanoma. Comparative analysis of biopsies revealed a higher fraction of ANGPT2-expressing tumor cells in metastatic versus primary sites. Tumor cell-expressed Angpt2 was dispensable for primary tumor growth, yet in-depth analysis of primary tumors revealed enhanced intratumoral necrosis upon silencing of tumor cell Angpt2 expression in the absence of significant immune and vascular alterations. Global transcriptional profiling of Angpt2-deficient tumor cells identified perturbations in redox homeostasis and an increased response to cellular oxidative stress. Ultrastructural analyses illustrated a significant increase of dysfunctional mitochondria in Angpt2-silenced tumor cells, thereby resulting in enhanced reactive oxygen species (ROS) production and downstream MAPK stress signaling. Functionally, enhanced ROS in Angpt2-silenced tumor cells reduced colonization potential in vitro and in vivo. Taken together, these findings uncover the hitherto unappreciated role of tumor cell-expressed ANGPT2 as an autocrine-positive regulator of metastatic colonization and validate ANGPT2 as a therapeutic target for a well-defined subset of patients with melanoma. SIGNIFICANCE: This study reveals that tumor cells can be a source of ANGPT2 in the tumor microenvironment and that tumor cell-derived ANGPT2 augments metastatic colonization by protecting tumor cells from oxidative stress.
Assuntos
Angiopoietina-2/metabolismo , Melanoma/secundário , Nevo/patologia , Neoplasias Cutâneas/patologia , Angiopoietina-2/genética , Animais , Comunicação Autócrina , Biópsia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Estimativa de Kaplan-Meier , Sistema de Sinalização das MAP Quinases , Melanoma/mortalidade , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Pele/patologia , Neoplasias Cutâneas/mortalidade , Análise Serial de Tecidos , Microambiente TumoralRESUMO
Tie1 is a mechanistically poorly characterized endothelial cell (EC)-specific orphan receptor. Yet, Tie1 deletion is embryonic lethal and Tie1 has been implicated in critical vascular pathologies, including atherosclerosis and tumor angiogenesis. Here, we show that Tie1 does not function independently but exerts context-dependent effects on the related receptor Tie2. Tie1 was identified as an EC activation marker that is expressed during angiogenesis by a subset of angiogenic tip and remodeling stalk cells and downregulated in the adult quiescent vasculature. Functionally, Tie1 expression by angiogenic EC contributes to shaping the tip cell phenotype by negatively regulating Tie2 surface presentation. In contrast, Tie1 acts in remodeling stalk cells cooperatively to sustain Tie2 signaling. Collectively, our data support an interactive model of Tie1 and Tie2 function, in which dynamically regulated Tie1 versus Tie2 expression determines the net positive or negative effect of Tie1 on Tie2 signaling.
Assuntos
Receptor de TIE-1/fisiologia , Receptor TIE-2/fisiologia , Remodelação Vascular/fisiologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Células Endoteliais/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologia , Receptor de TIE-1/genética , Receptor de TIE-1/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Vasos Retinianos/fisiologia , Transdução de SinaisRESUMO
Liver regeneration requires spatially and temporally precisely coordinated proliferation of the two major hepatic cell populations, hepatocytes and liver sinusoidal endothelial cells (LSECs), to reconstitute liver structure and function. The underlying mechanisms of this complex molecular cross-talk remain elusive. Here, we show that the expression of Angiopoietin-2 (Ang2) in LSECs is dynamically regulated after partial hepatectomy. During the early inductive phase of liver regeneration, Ang2 down-regulation leads to reduced LSEC transforming growth factor-ß1 production, enabling hepatocyte proliferation by releasing an angiocrine proliferative brake. During the later angiogenic phase of liver regeneration, recovery of endothelial Ang2 expression enables regenerative angiogenesis by controlling LSEC vascular endothelial growth factor receptor 2 expression. The data establish LSECs as a dynamic rheostat of liver regeneration, spatiotemporally orchestrating hepatocyte and LSEC proliferation through angiocrine- and autocrine-acting Ang2, respectively.
Assuntos
Angiopoietina-2/metabolismo , Proliferação de Células , Endotélio Vascular/metabolismo , Hepatócitos/fisiologia , Regeneração Hepática/fisiologia , Angiopoietina-2/genética , Animais , Hepatectomia , Hepatócitos/citologia , Regeneração Hepática/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Angiopoietin-2 (ANG-2) is a key regulator of angiogenesis that exerts context-dependent effects on ECs. ANG-2 binds the endothelial-specific receptor tyrosine kinase 2 (TIE2) and acts as a negative regulator of ANG-1/TIE2 signaling during angiogenesis, thereby controlling the responsiveness of ECs to exogenous cytokines. Recent data from tumors indicate that under certain conditions ANG-2 can also promote angiogenesis. However, the molecular mechanisms of dual ANG-2 functions are poorly understood. Here, we identify a model for the opposing roles of ANG-2 in angiogenesis. We found that angiogenesis-activated endothelium harbored a subpopulation of TIE2-negative ECs (TIE2lo). TIE2 expression was downregulated in angiogenic ECs, which abundantly expressed several integrins. ANG-2 bound to these integrins in TIE2lo ECs, subsequently inducing, in a TIE2-independent manner, phosphorylation of the integrin adaptor protein FAK, resulting in RAC1 activation, migration, and sprouting angiogenesis. Correspondingly, in vivo ANG-2 blockade interfered with integrin signaling and inhibited FAK phosphorylation and sprouting angiogenesis of TIE2lo ECs. These data establish a contextual model whereby differential TIE2 and integrin expression, binding, and activation control the role of ANG-2 in angiogenesis. The results of this study have immediate translational implications for the therapeutic exploitation of angiopoietin signaling.