RESUMO
BACKGROUND: Sex differences mediating predisposition to kidney injury are well known, with evidence indicating lower CKD incidence rates and slower decline in renal function in nondiabetic CKD for premenopausal women compared with men. However, signaling pathways involved have not been elucidated to date. The EGF receptor (EGFR) is widely expressed in the kidney in glomeruli and tubules, and persistent and dysregulated EGFR activation mediates progressive renal injury. METHODS: To investigate the sex differences in response to renal injury, we examined EGFR expression in mice, in human kidney tissue, and in cultured cell lines. RESULTS: In wild type mice, renal mRNA and protein EGFR levels were comparable in males and females at postnatal day 7 but were significantly lower in age-matched adult females than in adult males. Similar gender differences in renal EGFR expression were detected in normal adult human kidneys. In Dsk5 mutant mice with a gain-of-function allele that increases basal EGFR kinase activity, males had progressive glomerulopathy, albuminuria, loss of podocytes, and tubulointerstitial fibrosis, but female Dsk5 mice had minimal kidney injury. Oophorectomy had no effect on renal EGFR levels in female Dsk5 mice, while castration protected against the kidney injury in male Dsk5 mice, in association with a reduction in EGFR expression to levels seen in females. Conversely, testosterone increased EGFR expression and renal injury in female Dsk5 mice. Testosterone directly stimulated EGFR expression in cultured kidney cells. CONCLUSIONS: These studies indicate that differential renal EGFR expression plays a role in the sex differences in susceptibility to progressive kidney injury that may be mediated at least in part by testosterone.
Assuntos
Receptores ErbB/genética , Receptores ErbB/metabolismo , Rim/metabolismo , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Fatores Etários , Alelos , Animais , Castração , Linhagem Celular , Cloridrato de Erlotinib/farmacologia , Feminino , Mutação com Ganho de Função , Humanos , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Ovariectomia , Podócitos , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Insuficiência Renal Crônica/metabolismo , Fatores Sexuais , Testosterona/farmacologiaRESUMO
BACKGROUND: The discoidin domain receptor 1 (DDR1) is activated by collagens, upregulated in injured and fibrotic kidneys, and contributes to fibrosis by regulating extracellular matrix production, but how DDR1 controls fibrosis is poorly understood. DDR1 is a receptor tyrosine kinase (RTK). RTKs can translocate to the nucleus via a nuclear localization sequence (NLS) present on the receptor itself or a ligand it is bound to. In the nucleus, RTKs regulate gene expression by binding chromatin directly or by interacting with transcription factors. METHODS: To determine whether DDR1 translocates to the nucleus and whether this event is mediated by collagen-induced DDR1 activation, we generated renal cells expressing wild-type or mutant forms of DDR1 no longer able to bind collagen. Then, we determined the location of the DDR1 upon collagen stimulation. Using both biochemical assays and immunofluorescence, we analyzed the steps involved in DDR1 nuclear translocation. RESULTS: We show that although DDR1 and its natural ligand, collagen, lack an NLS, DDR1 is present in the nucleus of injured human and mouse kidney proximal tubules. We show that DDR1 nuclear translocation requires collagen-mediated receptor activation and interaction of DDR1 with SEC61B, a component of the Sec61 translocon, and nonmuscle myosin IIA and ß-actin. Once in the nucleus, DDR1 binds to chromatin to increase the transcription of collagen IV, a major collagen upregulated in fibrosis. CONCLUSIONS: These findings reveal a novel mechanism whereby activated DDR1 translates to the nucleus to regulate synthesis of profibrotic molecules.
Assuntos
Colágeno Tipo IV/genética , Colágeno Tipo I/metabolismo , Receptor com Domínio Discoidina 1/metabolismo , Túbulos Renais Proximais/metabolismo , Actinas/metabolismo , Injúria Renal Aguda/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Núcleo Celular , Cromatina/metabolismo , Colágeno Tipo I/farmacologia , Colágeno Tipo IV/metabolismo , Receptor com Domínio Discoidina 1/genética , Humanos , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Cadeias Pesadas de Miosina/metabolismo , Sinais de Localização Nuclear , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Canais de Translocação SEC/metabolismo , Transcrição GênicaRESUMO
The discovery of selective inhibitors of biological target proteins is the primary goal of many drug discovery campaigns. However, this goal has proven elusive, especially for inhibitors targeting the well-conserved orthosteric adenosine triphosphate (ATP) binding pocket of kinase enzymes. The human kinome is large and it is rather difficult to profile early lead compounds against around 500 targets to gain an upfront knowledge on selectivity. Further, selectivity can change drastically during derivatization of an initial lead compound. Here, we have introduced a computational model to support the profiling of compounds early in the drug discovery pipeline. On the basis of the extensive profiled activity of 70 kinase inhibitors against 379 kinases, including 81 tyrosine kinases, we developed a quantitative structure-activity relation (QSAR) model using artificial neural networks, to predict the activity of these kinase inhibitors against the panel of 379 kinases. The model's performance in predicting activity ranges from 0.6 to 0.8 depending on the kinase, from the area under the curve (AUC) of the receiver operating characteristics (ROC). The profiler is available online at http://www.meilerlab.org/index.php/servers/show?s_id=23.
Assuntos
Modelos Moleculares , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/química , Área Sob a Curva , Sítios de Ligação , Bases de Dados de Produtos Farmacêuticos , Descoberta de Drogas , Redes Neurais de Computação , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Quinases/química , Relação Quantitativa Estrutura-Atividade , Curva ROC , SoftwareRESUMO
Cell-extracellular matrix (ECM) interactions are essential for tissue development, homeostasis, and response to injury. Basement membranes (BMs) are specialized ECMs that separate epithelial or endothelial cells from stromal components and interact with cells via cellular receptors, including integrins and discoidin domain receptors. Disruption of cell-BM interactions due to either injury or genetic defects in either the ECM components or cellular receptors often lead to irreversible tissue injury and loss of organ function. Animal models that lack specific BM components or receptors either globally or in selective tissues have been used to help with our understanding of the molecular mechanisms whereby cell-BM interactions regulate organ function in physiological and pathological conditions. We review recently published works on animal models that explore how cell-BM interactions regulate kidney homeostasis in both health and disease.
Assuntos
Membrana Basal/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Rim/citologia , Rim/patologia , Receptores de Superfície Celular/metabolismo , Animais , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Rim/metabolismo , Ligação ProteicaRESUMO
Glomerulosclerosis is characterized by excessive deposition of extracellular matrix within the glomeruli of the kidney, glomerular cell death, and subsequent loss of functional glomeruli. While in physiological situations the levels of extracellular matrix components are kept constant by a tight balance between formation and degradation, in the case of injury that results in fibrosis there is increased matrix deposition relative to its breakdown. Multiple factors control matrix synthesis and degradation, thus contributing to the development of glomerulosclerosis. This review focuses primarily on the role of cell-matrix interactions, which play a critical role in governing glomerular cell cues in both healthy and diseased kidneys. Cell-extracellular matrix interactions are made possible by various cellular receptors including integrins, discoidin domain receptors, and dystroglycan. Upon binding to a selective extracellular matrix protein, these receptors activate intracellular signaling pathways that can either downregulate or upregulate matrix synthesis and deposition. This, together with the observation that changes in the expression levels of matrix receptors have been documented in glomerular disease, clearly emphasizes the contribution of cell-matrix interactions in glomerular injury. Understanding the molecular mechanisms whereby extracellular matrix receptors regulate matrix homeostasis in the course of glomerular injury is therefore critical for devising more effective therapies to treat and ideally prevent glomerulosclerosis.
Assuntos
Matriz Extracelular/metabolismo , Glomerulonefrite/metabolismo , Animais , Glomerulonefrite/patologia , Humanos , Integrinas/metabolismo , Rim/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologiaRESUMO
Mesangial cells and podocytes express integrins α1ß1 and α2ß1, which are the two major collagen receptors that regulate multiple cellular functions, including extracellular matrix homeostasis. Integrin α1ß1 protects from glomerular injury by negatively regulating collagen production, but the role of integrin α2ß1 in renal injury is unclear. Here, we subjected wild-type and integrin α2-null mice to injury with adriamycin or partial renal ablation. In both of these models, integrin α2-null mice developed significantly less proteinuria and glomerulosclerosis. In addition, selective pharmacological inhibition of integrin α2ß1 significantly reduced adriamycin-induced proteinuria, glomerular injury, and collagen deposition in wild-type mice. This inhibitor significantly reduced collagen synthesis in wild-type, but not integrin α2-null, mesangial cells in vitro, demonstrating that its effects are integrin α2ß1-dependent. Taken together, these results indicate that integrin α2ß1 contributes to glomerular injury by positively regulating collagen synthesis and suggest that its inhibition may be a promising strategy to reduce glomerular injury and proteinuria.
Assuntos
Injúria Renal Aguda/patologia , Doxorrubicina/farmacologia , Integrina alfa2beta1/metabolismo , Glomérulos Renais/lesões , Injúria Renal Aguda/metabolismo , Albuminúria/fisiopatologia , Análise de Variância , Animais , Western Blotting , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imuno-Histoquímica , Integrina alfa2beta1/efeitos dos fármacos , Testes de Função Renal , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Distribuição Aleatória , Receptores de Colágeno/metabolismoRESUMO
Following injury the kidney undergoes a repair process, which results in replacement of the injured tissue with little evidence of damage. However, repetitive injuries or inability of the kidney to stop the repair process result in abnormal deposition of extracellular matrix (ECM) components leading to fibrosis and organ dysfunction. The synthesis/degradation of ECM components is finely regulated by several factors, including discoidin domain receptors (DDRs). These are receptor tyrosine kinases that are activated by collagens. Upon activation, DDRs control several cell functions that, when exacerbated, contribute to kidney injury and fibrosis. DDRs are undetectable in healthy kidney, but become rapidly upregulated in several kidney fibrotic conditions, thus making them attractive anti-fibrotic targets. DDRs contribute to kidney injury and fibrosis by promoting apoptosis of injured kidney cells, stimulating the production of pro-inflammatory cytokines, and regulating the production of ECM components. They achieve these effects by activating canonical intracellular molecules or by directly interacting with nuclear chromatin and promoting the transcription of pro-fibrotic genes. The goal of this review is to highlight canonical and non-canonical mechanisms whereby DDRs contribute to kidney injury/fibrosis. This review will summarize key findings obtained using cells and mice lacking DDRs and it will discuss the discovery and development of targeted DDR small molecule- and antisense-based inhibitors. Understanding the molecular mechanisms whereby DDRs control kidney injury and fibrosis might enable us to not only develop more selective and potent inhibitors, but to also determine when DDR inhibition needs to be achieved to prevent and/or halt the development of kidney fibrosis.
RESUMO
Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase activated by collagen, contributes to chronic kidney disease. However, its role in acute kidney injury and subsequent development of kidney fibrosis is not clear. Thus, we performed a model of severe ischemia/reperfusion-induced acute kidney injury that progressed to kidney fibrosis in WT and Ddr1-null mice. We showed that Ddr1-null mice had reduced acute tubular injury, inflammation, and tubulointerstitial fibrosis with overall decreased renal monocyte chemoattractant protein (MCP-1) levels and STAT3 activation. We identified breakpoint cluster region (BCR) protein as a phosphorylated target of DDR1 that controls MCP-1 production in renal proximal tubule epithelial cells. DDR1-induced BCR phosphorylation or BCR downregulation increased MCP-1 secretion, suggesting that BCR negatively regulates the levels of MCP-1. Mechanistically, phosphorylation or downregulation of BCR increased ß-catenin activity and in turn MCP-1 production. Finally, we showed that DDR1-mediated STAT3 activation was required to stimulate the secretion of TGF-ß. Thus, DDR1 contributes to acute and chronic kidney injury by regulating BCR and STAT3 phosphorylation and in turn the production of MCP-1 and TGF-ß. These findings identify DDR1 an attractive therapeutic target for ameliorating both proinflammatory and profibrotic signaling in kidney disease.
Assuntos
Receptor com Domínio Discoidina 1/genética , Regulação da Expressão Gênica , Inflamação/complicações , Túbulos Renais Proximais/metabolismo , Proteínas Proto-Oncogênicas c-bcr/genética , RNA/genética , Fator de Transcrição STAT3/genética , Injúria Renal Aguda , Animais , Linhagem Celular , Células Cultivadas , Receptor com Domínio Discoidina 1/biossíntese , Feminino , Fibrose/complicações , Fibrose/genética , Fibrose/patologia , Inflamação/genética , Inflamação/patologia , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Proteínas Proto-Oncogênicas c-bcr/biossíntese , Fator de Transcrição STAT3/biossíntese , Transdução de SinaisRESUMO
Integrins and discoidin domain receptors (DDRs) 1 and 2 promote cell adhesion and migration on both fibrillar and non fibrillar collagens. Collagen I contains DDR and integrin selective binding motifs; however, the relative contribution of these two receptors in regulating cell migration is unclear. DDR1 has five isoforms (DDR1a-e), with most cells expressing the DDR1a and DDR1b isoforms. We show that human embryonic kidney 293 cells expressing DDR1b migrate more than DDR1a expressing cells on DDR selective substrata as well as on collagen I in vitro. In addition, DDR1b expressing cells show increased lung colonization after tail vein injection in nude mice. DDR1a and DDR1b differ from each other by an extra 37 amino acids in the DDR1b cytoplasmic domain. Interestingly, these 37 amino acids contain an NPxY motif which is a central control module within the cytoplasmic domain of ß integrins and acts by binding scaffold proteins, including talin. Using purified recombinant DDR1 cytoplasmic tail proteins, we show that DDR1b directly binds talin with higher affinity than DDR1a. In cells, DDR1b, but not DDR1a, colocalizes with talin and integrin ß1 to focal adhesions and enhances integrin ß1-mediated cell migration. Moreover, we show that DDR1b promotes cell migration by enhancing Rac1 activation. Mechanistically DDR1b interacts with the GTPase-activating protein (GAP) Breakpoint cluster region protein (BCR) thus reducing its GAP activity and enhancing Rac activation. Our study identifies DDR1b as a major driver of cell migration and talin and BCR as key players in the interplay between integrins and DDR1b in regulating cell migration.
RESUMO
Integrin α1ß1 is a collagen receptor that down-regulates collagen and reactive oxygen species (ROS) production, and mice lacking this receptor show increased ROS levels and exacerbated glomerular sclerosis following injury. Caveolin-1 (Cav-1) is a multifunctional protein that is tyrosine-phosphorylated in response to injury and has been implicated in ROS-mediated injury. Cav-1 interacts with integrins, and integrin α1ß1 binds/activates T cell protein-tyrosine phosphatase (TCPTP), which is homologous to the tyrosine phosphatase PTP1B known to dephosphorylate Cav-1. In this study, we analyzed whether phosphorylated Cav-1 (pCav-1) is a substrate of TCPTP and if integrin α1ß1 is essential for promoting TCPTP-mediated Cav-1 dephosphorylation. We found that Cav-1 phosphorylation is significantly higher in cells lacking integrin α1ß1 at base line and following oxidative stress. Overexpression of TCPTP leads to reduced pCav-1 levels only in cells expressing integrin α1ß1. Using solid phase binding assays, we demonstrated that 1) purified Cav-1 directly interacts with TCPTP and the integrin α1 subunit, 2) pCav-1 is a substrate of TCPTP, and 3) TCPTP-mediated Cav-1 dephosphorylation is highly increased by the addition of purified integrin α1ß1 or an integrin α1 cytoplasmic peptide to which TCPTP has been shown to bind. Thus, our results demonstrate that pCav-1 is a new substrate of TCPTP and that integrin α1ß1 acts as a negative regulator of Cav-1 phosphorylation by activating TCPTP. This could explain the protective function of integrin α1ß1 in oxidative stress-mediated damage and why integrin α1-null mice are more susceptible to fibrosis following injury.
Assuntos
Caveolina 1/metabolismo , Integrina alfa1beta1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Animais , Células CHO , Caveolina 1/genética , Colágeno/genética , Colágeno/metabolismo , Cricetinae , Cricetulus , Ativação Enzimática/genética , Fibrose/genética , Células HEK293 , Humanos , Integrina alfa1beta1/genética , Camundongos , Camundongos Mutantes , Estresse Oxidativo/genética , Fosforilação/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Epstein-Barr virus is ubiquitous and is causally implicated in lymphoid and epithelial malignancies. Virus invades oropharyngeal mucosa and establishes latency in B lymphocytes. Reactivating lymphocytes shed virus into saliva for spread to new hosts. A complex of three virus glycoproteins, gH, gL and gp42, is essential for entry. B-cell entry requires binding of gp42 to human leukocyte antigen (HLA) class II whereas entry into epithelial cells lacking HLA class II requires complexes without gp42. To accommodate infection of each, the virus carries both three-part and two-part complexes. We show here that HLA class II in the virus-producing cell alters the ratio of three-part to two-part complexes. As a consequence, virus originating in epithelial cells efficiently infects B cells whereas B-cell derived virus better infects epithelial cells. This molecular switch is a novel strategy that could alter tropism of virus from epithelium to B cells and then back to epithelium in a new host.
Assuntos
Linfócitos B/virologia , Células Epiteliais/virologia , Herpesvirus Humano 4/fisiologia , Replicação Viral/fisiologia , Animais , Linfócitos B/imunologia , Linhagem Celular , DNA Viral/análise , Células Epiteliais/imunologia , Deleção de Genes , Herpesvirus Humano 4/genética , Antígenos de Histocompatibilidade Classe II/análise , Ativação Linfocitária , Timidina Quinase/deficiência , Timidina Quinase/genética , Eliminação de Partículas ViraisRESUMO
Integrin alpha1beta1, the major collagen type IV receptor, is expressed by endothelial cells and plays a role in both physiologic and pathologic angiogenesis. Because the molecular mechanisms whereby this collagen IV receptor mediates endothelial cell functions are poorly understood, truncation and point mutants of the integrin alpha1 subunit cytoplasmic tail (amino acids 1137-1151) were generated and expressed into alpha1-null endothelial cells. We show that alpha1-null endothelial cells expressing the alpha1 subunit, which lacks the entire cytoplasmic tail (mutant alpha1-1136) or expresses all the amino acids up to the highly conserved GFFKR motif (mutant alpha1-1143), have a similar phenotype to parental alpha1-null cells. Pro(1144) and Leu(1145) were shown to be necessary for alpha1beta1-mediated endothelial cell proliferation; Lys(1146) for adhesion, migration, and tubulogenesis and Lys(1147) for tubulogenesis. Integrin alpha1beta1-dependent endothelial cell proliferation is primarily mediated by ERK activation, whereas migration and tubulogenesis require both p38 MAPK and PI3K/Akt activation. Thus, distinct amino acids distal to the GFFKR motif of the alpha1 integrin cytoplasmic tail mediate activation of selective downstream signaling pathways and specific endothelial cell functions.
Assuntos
Citoplasma/metabolismo , Células Endoteliais/metabolismo , Integrina alfa1/química , Motivos de Aminoácidos , Adesão Celular , Movimento Celular , Proliferação de Células , Humanos , Mutação , Neovascularização Patológica , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Mutação Puntual , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Diabetic nephropathy (DN) affects both glomerular cells and the extracellular matrix (ECM), yet the pathogenic mechanisms involving cell-matrix interactions are poorly understood. Glycation alters integrin-dependent cell-ECM interactions, and perturbation of these interactions results in severe renal pathology in diabetic animals. Here, we investigated how chemical modifications of the ECM by hyperglycemia and carbonyl stress, two major features of the diabetic milieu, affect mesangial cell functions. Incubation of collagen IV with pathophysiological levels of either the carbonyl compound methylglyoxal (MGO) or glucose resulted in modification of arginine or lysine residues, respectively. Mouse mesangial cells plated on MGO-modified collagen IV showed decreased adhesion and migration. Cells plated on glucose-modified collagen IV showed reduced proliferation and migration and increased collagen IV production. Inhibiting glucose-mediated oxidative modification of collagen IV lysine residues rescued the alterations in cell growth, migration, and collagen synthesis. We propose that diabetic ECM affects mesangial cell functions via two distinct mechanisms: modification of arginine residues by MGO inhibits cell adhesion, whereas oxidative modification of lysine residues by glucose inhibits cell proliferation and increases collagen IV production. These mechanisms may contribute to mesangial cell hypertrophy and matrix expansion in DN.
Assuntos
Colágeno Tipo IV/metabolismo , Nefropatias Diabéticas/etiologia , Glucose/toxicidade , Células Mesangiais/fisiologia , Aldeído Pirúvico/toxicidade , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , CamundongosRESUMO
Herein, we report the discovery of a potent and selective dual DDR1/2 inhibitor, 7e (VU6015929), displaying low cytotoxicity, good kinome selectivity, and possessing an acceptable in vitro DMPK profile with good rodent in vivo pharmacokinetics. VU6015929 potently blocks collagen-induced DDR1 activation and collagen-IV production, suggesting DDR1 inhibition as an exciting target for antifibrotic therapy.
RESUMO
Podocyte adhesion to the glomerular basement membrane is required for proper function of the glomerular filtration barrier. However, the mechanism whereby podocytes adhere to collagen IV networks, a major component of the glomerular basement membrane, is poorly understood. The predominant collagen IV network is composed of triple helical protomers containing the alpha3alpha4alpha5 chains. The protomers connect via the trimeric noncollagenous (NC1) domains to form hexamers at the interface. Because the NC1 domains of this network can potentially support integrin-dependent cell adhesion, it was determined whether individual NC1 monomers or alpha3alpha4alpha5 hexamers support podocyte adhesion. It was found that, although human podocytes did not adhere to NC1 domains proper, they did adhere via integrin alphavbeta3 to a KRGDS motif located adjacent to alpha3NC1 domains. Because the KRGDS motif is a site of phosphorylation, its interactions with integrin alphavbeta3 may play a critical role in cell signaling in physiologic and pathologic states.
Assuntos
Colágeno Tipo IV , Podócitos/fisiologia , Motivos de Aminoácidos , Animais , Adesão Celular , HumanosRESUMO
Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds to and is activated by collagens. DDR1 expression increases following kidney injury and accumulating evidence suggests that it contributes to the progression of injury. To this end, deletion of DDR1 is beneficial in ameliorating kidney injury induced by angiotensin infusion, unilateral ureteral obstruction, or nephrotoxic nephritis. Most of the beneficial effects observed in the DDR1-null mice are attributed to reduced inflammatory cell infiltration to the site of injury, suggesting that DDR1 plays a pro-inflammatory effect. The goal of this study was to determine whether, in addition to its pro-inflammatory effect, DDR1 plays a deleterious effect in kidney injury by directly regulating extracellular matrix production. We show that DDR1-null mice have reduced deposition of glomerular collagens I and IV as well as decreased proteinuria following the partial renal ablation model of kidney injury. Using mesangial cells isolated from DDR1-null mice, we show that these cells produce significantly less collagen compared to DDR1-null cells reconstituted with wild type DDR1. Moreover, mutagenesis analysis revealed that mutations in the collagen binding site or in the kinase domain significantly reduce DDR1-mediated collagen production. Finally, we provide evidence that blocking DDR1 kinase activity with an ATP-competitive small molecule inhibitor reduces collagen production. In conclusion, our studies indicate that the kinase activity of DDR1 plays a key role in DDR1-induced collagen synthesis and suggest that blocking collagen-mediated DDR1 activation may be beneficial in fibrotic diseases.
Assuntos
Injúria Renal Aguda/genética , Colágeno Tipo IV/genética , Receptor com Domínio Discoidina 1/genética , Glomérulos Renais/metabolismo , Nefrite/genética , Obstrução Ureteral/metabolismo , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Injúria Renal Aguda/cirurgia , Angiotensinas , Animais , Sítios de Ligação , Colágeno Tipo IV/metabolismo , Receptor com Domínio Discoidina 1/deficiência , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica , Humanos , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Knockout , Nefrectomia , Nefrite/induzido quimicamente , Nefrite/metabolismo , Nefrite/patologia , Ligação Proteica , Transdução de Sinais , Ureter/cirurgia , Obstrução Ureteral/patologia , Obstrução Ureteral/cirurgiaRESUMO
Discoidin domain receptor (DDR) 1 and 2 are transmembrane receptors that belong to the family of receptor tyrosine kinases (RTK). Upon collagen binding, DDRs transduce cellular signaling involved in various cell functions, including cell adhesion, proliferation, differentiation, migration, and matrix homeostasis. Altered DDR function resulting from either mutations or overexpression has been implicated in several types of disease, including atherosclerosis, inflammation, cancer, and tissue fibrosis. Several established inhibitors, such as imatinib, dasatinib, and nilotinib, originally developed as Abelson murine leukemia (Abl) kinase inhibitors, have been found to inhibit DDR kinase activity. As we review here, recent discoveries of novel inhibitors and their co-crystal structure with the DDR1 kinase domain have made structure-based drug discovery for DDR1 amenable.
Assuntos
Descoberta de Drogas , Receptores Proteína Tirosina Quinases/química , Receptores Mitogênicos/química , Animais , Receptores com Domínio Discoidina , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Mitogênicos/antagonistas & inibidoresRESUMO
Discoidin domain receptors, DDR1 and DDR2, lie at the intersection of two large receptor families, namely the extracellular matrix and tyrosine kinase receptors. As such, DDRs are uniquely positioned to function as sensors for extracellular matrix and to regulate a wide range of cell functions from migration and proliferation to cytokine secretion and extracellular matrix homeostasis/remodeling. While activation of DDRs by extracellular matrix collagens is required for normal development and tissue homeostasis, aberrant activation of these receptors following injury or in disease is detrimental. The availability of mice lacking DDRs has enabled us to identify key roles played by these receptors in disease initiation and progression. DDR1 promotes inflammation in atherosclerosis, lung fibrosis and kidney injury, while DDR2 contributes to osteoarthritis. Furthermore, both DDRs have been implicated in cancer progression. Yet the mechanisms whereby DDRs contribute to disease progression are poorly understood. In this review we highlight the mechanisms whereby DDRs regulate two important processes, namely inflammation and tissue fibrosis. In addition, we discuss the challenges of targeting DDRs in disease. Selective targeting of these receptors requires understanding of how they interact with and are activated by extracellular matrix, and whether their cellular function is dependent on or independent of receptor kinase activity.
Assuntos
Fibrose/genética , Inflamação/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Mitogênicos/genética , Animais , Movimento Celular/genética , Proliferação de Células , Receptor com Domínio Discoidina 1 , Receptores com Domínio Discoidina , Matriz Extracelular/genética , Matriz Extracelular/patologia , Fibrose/patologia , Humanos , Inflamação/patologia , Camundongos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Mitogênicos/metabolismo , Transdução de Sinais/genéticaRESUMO
Integrin alpha1beta1 negatively regulates the generation of profibrotic reactive oxygen species (ROS) by inhibiting epidermal growth factor receptor (EGFR) activation; however, the mechanism by which it does this is unknown. In this study, we show that caveolin-1 (Cav-1), a scaffolding protein that binds integrins and controls growth factor receptor signaling, participates in integrin alpha1beta1-mediated EGFR activation. Integrin alpha1-null mesangial cells (MCs) have reduced Cav-1 levels, and reexpression of the integrin alpha1 subunit increases Cav-1 levels, decreases EGFR activation, and reduces ROS production. Downregulation of Cav-1 in wild-type MCs increases EGFR phosphorylation and ROS synthesis, while overexpression of Cav-1 in the integrin alpha1-null MCs decreases EGFR-mediated ROS production. We further show that integrin alpha1-null MCs have increased levels of activated extracellular signal-regulated kinase (ERK), which leads to reduced activation of peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor that positively regulates Cav-1 expression. Moreover, activation of PPARgamma or inhibition of ERK increases Cav-1 levels in the integrin alpha1-null MCs. Finally, we show that glomeruli of integrin alpha1-null mice have reduced levels of Cav-1 and activated PPARgamma but increased levels of phosphorylated EGFR both at baseline and following injury. Thus, integrin alpha1beta1 negatively regulates EGFR activation by positively controlling Cav-1 levels, and the ERK/PPARgamma axis plays a key role in regulating integrin alpha1beta1-dependent Cav-1 expression and consequent EGFR-mediated ROS production.