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OBJECTIVE: Mitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized. METHODS: We studied two model systems for HSP60 deficiency: (1) engineered HEK cells carrying an inducible dominant negative HSP60 mutant protein, (2) zebrafish HSP60 knockout larvae. Both systems were analyzed by RNASeq, proteomics, and targeted metabolomics, and several functional assays relevant for the respective model. In addition, skin fibroblasts from patients with disease-associated HSP60 variants were analyzed by proteomics. RESULTS: We show that HSP60 deficiency leads to a differentially downregulated mitochondrial matrix proteome, transcriptional activation of stress responses, and dysregulated cholesterol biosynthesis. This leads to lipid accumulation in zebrafish knockout larvae. CONCLUSIONS: Our data provide a compendium of the effects of HSP60 deficiency on the mitochondrial matrix proteome. We show that HSP60 is a master regulator and modulator of mitochondrial functions and metabolic pathways. HSP60 dysfunction also affects cellular metabolism and disrupts the integrated stress response. The effect on cholesterol synthesis explains the effect of HSP60 dysfunction on myelination observed in patients carrying genetic variants of HSP60.
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INTRODUCTION: Insulin-like growth factor (IGF)1 and IGF2 have neuroprotective effects, but less is known regarding how other members of the IGF system, including IGF binding proteins (IGFBPs) and the regulatory proteinase pappalysin-1 (PAPP-A) and its endogenous inhibitor stanniocalcin-2 (STC2) participate in this process. Here, we analyzed whether these members of the IGF system are modified in neurons and astrocytes in response to palmitic acid (PA), a fatty acid that induces cell stress when increased centrally. METHODS: Primary hypothalamic astrocyte cultures from male and female PND2 rats and the pro-opiomelanocortin (POMC) neuronal cell line, mHypoA-POMC/GFP-2, were treated with PA, IGF1 or both. To analyze the role of STC2 in astrocytes, siRNA assays were employed. RESULTS: In astrocytes of both sexes, PA rapidly increased cell stress factors followed by increased Pappa and Stc2 mRNA levels and then a decrease in Igf1, Igf2, and Igfbp2 expression and cell number. Exogenous IGF1 did not revert these effects. In mHypoA-POMC/GFP-2 neurons, PA reduced cell number and Pomc and Igf1 mRNA levels, and increased Igfbp2 and Stc2, again with no effect of exogenous IGF1. PA increased STC2 expression, but no effects of decreasing its levels by interference assays or exogenous STC2 treatment in astrocytes were found. CONCLUSIONS: The response of the IGF system to PA was cell and sex specific, but no protective effects of the IGFs were found. However, the modifications in hypothalamic PAPP-A and STC2 indicate that further studies are required to determine their role in the response to fatty acids and possibly in metabolic control.
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Zebrafish have become a widely used vertebrate model in physiology and reliable measures of their metabolic rate are needed. We have developed a 3D-printed respirometer and swim tunnel system and used it for obtaining accurate measurement of standard metabolic rate (SMR) and maximal, aerobic metabolic rate (MMR) in zebrafish under rest and maximal exercise, respectively. We compared a slow (stepwise) protocol to a fast (continuous) protocol for determining MMR. The fast protocol yielded slightly (but not significantly) higher oxygen consumption rates than the slow protocol and the data, in contrast to the slow protocol, followed a normal distribution. These findings point to the fast protocol as a fast and reliable method for obtaining accurate values of MMR in zebrafish. We make the 3D drawings for printing the system available to researchers, to help streamline the field of metabolic research in zebrafish and other smaller fish species.
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Consumo de Oxígeno , Impresión Tridimensional , Natación , Pez Cebra , Animales , Metabolismo Energético , Metabolismo BasalRESUMEN
BACKGROUND: Prepubertal children with obesity frequently have enhanced growth, accelerated skeletal maturation and changes in the GH-IGF axis. However, the involvement of pappalysins (PAPP-A, PAPP-A2) and stanniocalcins (STC1, STC2) as regulators of IGF bioavailability has not been studied in obesity. OBJECTIVE: We aimed to determine the effects of childhood obesity and weight reduction on serum levels of PAPP-A, PAPP-A2, STC1 and STC2 and their relationship with IGF bioavailability, growth, and other components of the GH-IGF system. PATIENTS AND METHODS: Prepubertal children with severe obesity (150, 50% males/females, age: 7.72 ± 2.05 years, BMI z-score: 4.95 ± 1.70, height z-score: 1.28 ± 1.04) were studied at diagnosis and after a minimum of 0.5 BMI z-score reduction. Two hundred and six healthy age- and sex-matched children were used as controls. RESULTS: Children with obesity had decreased serum concentrations of PAPP-A, PAPP-A2 and STC2, but increased total and free IGF-I (fIGF-I), intact IGFBP-3, ALS, IGF-II and insulin levels, with no difference in the free/total IGF-I ratio. Neither the standardized BMI nor height correlated with any biochemical parameter analyzed. A decrease in IGF-II, insulin, and ALS with an increase in IGFBP-2 and -5, STC2 and PAPP-A were observed after weight loss. CONCLUSION: Increased circulating total and free IGF-I, insulin and IGF-II may all contribute to the increased rate of prepubertal growth and bone maturation observed in children with obesity, with STC2 possibly being involved.
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CONTEXT: IGF signalling is known to affect human ovarian follicular function during growth and development. However, the role of the IGF system is unknown during the ovulatory peak, which is characterized by profound changes in granulosa cell (GCs) mitosis and function. OBJECTIVE: How is the IGF system expressed and regulated during the midcycle surge in women? DESIGN: Follicular fluid (FF) and granulosa cells (GCs) were collected during the ovulatory peak from two specific time-points. One sample was obtained before oocyte pick up (OPU): before ovulation trigger (OT) (T = 0 h) or at 12, 17, or 32 h after OT, and one sample was obtained at OPU 36 h after OT. SETTING: University hospital. PATIENTS/PARTICIPANTS: Fifty women undergoing ovarian stimulation were included. MAIN OUTCOME MEASURE: Gene expression profiles were assessed by microarray analysis of GCs. IGF-related proteins in the FF were assessed by using immunoassays or by determination of activity with a proteinase assay. RESULTS: Expression of proteins promoting IGF activity (i.e., IGF2, PAPPA, and IRS1) together with proliferation markers were downregulated on a transcriptional level in GCs after OT, whereas proteins inhibiting the IGF signal (i.e., IGFBPs, IGF2R, and STC1) were upregulated. STC1 gene expression and protein levels were greatly upregulated after OT with a parallel steep downregulation of PAPP-A proteolytic activity. CONCLUSIONS: These data suggest that downregulation of IGF signalling mediated by increased STC1 expression is instrumental for the sudden cessation in GC proliferation and onset of differentiation during the ovulatory peak.
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The pappalysins pregnancy associated plasma protein-A (PAPP-A) and -A2 (PAPP-A2) act as proteinases of insulin-like growth factor-1 (IGF-1) binding proteins, while stanniocalcin-2 (STC2) was identified as a pappalysin inhibitor. While there is some evidence from studies in children and adolescents, it is unclear whether these molecules are related to concentrations of IGF-1 and its binding proteins in adults. We investigated cross-sectionally the association of circulating PAPP-A, PAPP-A2 and STC2 with IGF-1 and its binding proteins (IGFBPs) in 394 adult pretest participants (20-69 years) of the German National Cohort Berlin North study center. Plasma PAPP-A, PAPP-A2, total and free IGF-1, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-5 and STC2 were measured by ELISAs. The associations of PAPP-A, PAPP-A2 and STC2 with IGF-1 or IGFBPs were investigated using multivariable linear regression analyses adjusting for age, sex, body mass index and pretest phase. We observed significant inverse associations of PAPP-A2 (difference in concentrations per 0.5 ng/mL higher PAPP-A2 levels) with total IGF-1 (- 4.3 ng/mL; 95% CI - 7.0; - 1.6), the IGF-1:IGFBP-3 molar ratio (- 0.34%; 95%-CI - 0.59; - 0.09), but not free IGF-1 and a positive association with IGFBP-2 (11.9 ng/mL; 95% CI 5.0; 18.8). PAPP-A was not related to total or free IGF-1, but positively associated with IGFBP-5. STC2 was inversely related to total IGF-1, IGFBP-2 and IGFBP-3 and positively to IGFBP-1. This first investigation of these associations in a general adult population supports the hypothesis that PAPP-A2 as well as STC2 play a role for IGF-1 and its binding proteins, especially for total IGF-1. The role of PAPP-A2 and STC2 for health and disease in adults warrants further investigation.
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Factor I del Crecimiento Similar a la Insulina , Hormonas Peptídicas , Piperazinas , Adulto , Humanos , Proteínas Portadoras , Glicoproteínas/metabolismo , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina , Factor I del Crecimiento Similar a la Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Hormonas Peptídicas/metabolismo , Proteína Plasmática A Asociada al Embarazo/metabolismo , Adulto Joven , Persona de Mediana Edad , AncianoRESUMEN
Motor neuron (MN) development and nerve regeneration requires orchestrated action of a vast number of molecules. Here, we identify SorCS2 as a progranulin (PGRN) receptor that is required for MN diversification and axon outgrowth in zebrafish and mice. In zebrafish, SorCS2 knockdown also affects neuromuscular junction morphology and fish motility. In mice, SorCS2 and PGRN are co-expressed by newborn MNs from embryonic day 9.5 until adulthood. Using cell-fate tracing and nerve segmentation, we find that SorCS2 deficiency perturbs cell-fate decisions of brachial MNs accompanied by innervation deficits of posterior nerves. Additionally, adult SorCS2 knockout mice display slower motor nerve regeneration. Interestingly, primitive macrophages express high levels of PGRN, and their interaction with SorCS2-positive motor axon is required during axon pathfinding. We further show that SorCS2 binds PGRN to control its secretion, signaling, and conversion into granulins. We propose that PGRN-SorCS2 signaling controls MN development and regeneration in vertebrates.
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Péptidos y Proteínas de Señalización Intercelular , Pez Cebra , Ratones , Animales , Progranulinas , Pez Cebra/metabolismo , Neuronas Motoras/metabolismo , Granulinas , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Previous studies using myoglobin (Mb) knockout mice and knockdown zebrafish have presented conflicting results about in vivo phenotypes resulting from the loss of this conserved and highly expressed protein, and therefore a new well-characterized knockout model is warranted. We here describe the generation of three distinct zebrafish mb knockout lines using the CRISPR/Cas system. None of the three lines exhibited any morphological phenotypes, changes in length, or lethality during embryonic and larval development. The adult homozygous knockout mb(Auzf13.2) zebrafish line were absent of Mb protein, had an almost complete degradation of mb mRNA, and showed no changes in viability, length, or heart size. Furthermore, transcriptomic analysis of adult heart tissue showed that mb knockout did not cause altered expression of other genes. Lastly, no off-targeting was observed in 36 screened loci. In conclusion, we have generated three mb knockout lines with indistinguishable phenotypes during embryonic and larval development and validated one of these lines, mb(Auzf13.2), to have no signs of genetic compensation or off-target effects in the adult heart. These findings suggests that the mb(Auzf13.2) shows promise as a candidate for investigating the biological role of Mb in zebrafish.
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Mioglobina , Pez Cebra , Animales , Ratones , Pez Cebra/genética , Pez Cebra/metabolismo , Mioglobina/genética , Mioglobina/metabolismo , Proteínas de Pez Cebra/genética , Sistemas CRISPR-Cas , Fenotipo , Técnicas de Inactivación de GenesRESUMEN
AIMS: Stanniocalcin-2 (STC2) has recently been implicated in human muscle mass variability by genetic analysis. Biochemically, STC2 inhibits the proteolytic activity of the metalloproteinase PAPP-A, which promotes muscle growth by upregulating the insulin-like growth factor (IGF) axis. The aim was to examine if STC2 affects skeletal muscle mass and to assess how the IGF axis mediates muscle hypertrophy induced by functional overload. METHODS: We compared muscle mass and muscle fiber morphology between Stc2-/- (n = 21) and wild-type (n = 15) mice. We then quantified IGF1, IGF2, IGF binding proteins -4 and -5 (IGFBP-4, IGFBP-5), PAPP-A and STC2 in plantaris muscles of wild-type mice subjected to 4-week unilateral overload (n = 14). RESULTS: Stc2-/- mice showed up to 10% larger muscle mass compared with wild-type mice. This increase was mediated by greater cross-sectional area of muscle fibers. Overload increased plantaris mass and components of the IGF axis, including quantities of IGF1 (by 2.41-fold, p = 0.0117), IGF2 (1.70-fold, p = 0.0461), IGFBP-4 (1.48-fold, p = 0.0268), PAPP-A (1.30-fold, p = 0.0154) and STC2 (1.28-fold, p = 0.019). CONCLUSION: Here we provide evidence that STC2 is an inhibitor of muscle growth upregulated, along with other components of the IGF axis, during overload-induced muscle hypertrophy.
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Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina , Hormonas Peptídicas , Animales , Ratones , Glicoproteínas/genética , Glicoproteínas/metabolismo , Hipertrofia , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Músculo Esquelético/metabolismo , Hormonas Peptídicas/metabolismo , Proteína Plasmática A Asociada al Embarazo/genéticaRESUMEN
The genetic architecture of the QT interval, defined as the period from onset of depolarisation to completion of repolarisation of the ventricular myocardium, is incompletely understood. Only a minor part of the QT interval variation in the general population has been linked to autosomal variant loci. Altered X chromosome dosage in humans, as seen in sex chromosome aneuploidies such as Turner syndrome (TS) and Klinefelter syndrome (KS), is associated with altered QTc interval (heart rate corrected QT), indicating that genes, located in the pseudoautosomal region 1 of the X and Y chromosomes may contribute to QT interval variation. We investigate the dosage effect of the pseudoautosomal gene SLC25A6, encoding the membrane ADP/ATP translocase 3 in the inner mitochondrial membrane, on QTc interval duration. To this end we used human participants and in vivo zebrafish models. Analyses in humans, based on 44 patients with KS, 44 patients with TS, 59 male and 22 females, revealed a significant negative correlation between SLC25A6 expression level and QTc interval duration. Similarly, downregulation of slc25a6 in zebrafish increased QTc interval duration with pharmacological inhibition of KATP channels restoring the systolic duration, whereas overexpression of SLC25A6 shortened QTc, which was normalized by pharmacological activation of KATP channels. Our study demonstrate an inverse relationship between SLC25A6 dosage and QTc interval indicating that SLC25A6 contributes to QT interval variation.
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Síndrome de Klinefelter , Síndrome de QT Prolongado , Síndrome de Turner , Animales , Femenino , Humanos , Masculino , Adenosina Trifosfato , Electrocardiografía , Síndrome de QT Prolongado/genética , Cromosoma X , Pez Cebra/genética , Translocador 3 del Nucleótido AdeninaRESUMEN
Pregnancy-associated plasma protein-A (PAPP-A) was first identified in the early 1970s as a placental protein of unknown function, present at high concentrations in the circulation of pregnant women. In the mid-to-late 1990s, PAPP-A was discovered to be a metzincin metalloproteinase, expressed by many nonplacental cells, that regulates local insulin-like growth factor (IGF) activity through cleavage of high-affinity IGF binding proteins (IGFBPs), in particular IGFBP-4. With PAPP-A as a cell surface-associated enzyme, the reduced affinity of the cleavage fragments results in increased IGF available to bind and activate IGF receptors in the pericellular environment. This proteolytic regulation of IGF activity is important, since the IGFs promote proliferation, differentiation, migration, and survival in various normal and cancer cells. Thus, there has been a steady growth in investigation of PAPP-A structure and function outside of pregnancy. This review provides historical perspective on the discovery of PAPP-A and its structure and cellular function, highlights key studies of the first 50 years in PAPP-A research, and introduces new findings from recent years.
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Placenta , Proteína Plasmática A Asociada al Embarazo , Embarazo , Humanos , Femenino , Metaloproteasas , Diferenciación Celular , Factor I del Crecimiento Similar a la InsulinaRESUMEN
BACKGROUND: A variant in the SLC4A3 anion exchanger has been identified as a novel cause of short QT syndrome (SQTS), but the clinical importance of SLC4A3 as a cause of SQTS or sudden cardiac death remains unknown. OBJECTIVE: The purpose of this study was to investigate the prevalence of potential disease-causing variants in SQTS patients using gene panels including SLC4A3. METHODS: In this multicenter study, genetic testing was performed in 34 index patients with SQTS. The pathogenicity of novel SLC4A3variants was validated in a zebrafish embryo heart model. RESULTS: Potentially disease-causing variants were identified in 9 (26%) patients and were mainly (15%) located in SLC4A3: 4 patients heterozygous for novel nonsynonymous SLC4A3 variants-p.Arg600Cys, p.Arg621Trp, p.Glu852Asp, and p.Arg952His-and 1 patient with the known p.Arg370His variant. In other SQTS genes, potentially disease-causing variants were less frequent (2× in KCNQ1, 1× in KCNJ2, and CACNA1C each). SLC4A3 variant carriers (n = 5) had a similar heart rate but shorter QT and J point to T wave peak intervals than did noncarriers (n = 29). Knockdown of slc4a3 in zebrafish resulted in shortened heart rate-corrected QT intervals (calculated using the Bazett formula) that could be rescued by overexpression of the native human SLC4A3-encoded protein (AE3), but neither by the mutated AE3 variants p.Arg600Cys, p.Arg621Trp, p.Glu852Asp nor by p.Arg952His, suggesting pathogenicity of these variants. Dysfunction in slc4a3/AE3 was associated with alkaline cytosol and shortened action potential of cardiomyocytes. CONCLUSION: In about a quarter of patients with SQTS, a potentially disease-causing variant can be identified. Nonsynonymous variants in SLC4A3 represent the most common cause of SQTS, underscoring the importance of including SLC4A3 in the genetic screening of patients with SQTS or sudden cardiac death.
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Electrocardiografía , Pez Cebra , Animales , Humanos , Arritmias Cardíacas , Muerte Súbita Cardíaca/prevención & control , Electrocardiografía/métodosRESUMEN
The pappalysin metalloproteinases, PAPP-A and PAPP-A2, have emerged as highly specific proteolytic enzymes involved in the regulation of insulin-like growth factor (IGF) signaling. The only known pappalysin substrates are a subset of the IGF binding proteins (IGFBPs), which bind IGF-I or IGF-II with high affinity to antagonize receptor binding. Thus, by cleaving IGFBPs, the pappalysins have the potential to increase IGF bioactivity and hence promote IGF signaling. This is relevant both in systemic and local IGF regulation, in normal and several pathophysiological conditions. Stanniocalcin-1 and -2 were recently found to be potent pappalysin inhibitors, thus comprising the missing components of a complete proteolytic system, the stanniocalcin-PAPP-A-IGFBP-IGF axis. Here, we provide the biological context necessary for understanding the properties of this molecular network, and we review biochemical data, animal experiments, clinical data, and genetic data supporting the physiological operation of this branch as an important part of the IGF system. However, although in vivo data clearly illustrate its power, it is a challenge to understand its subtle operation, for example, multiple equilibria and inhibitory kinetics may determine how, where, and when the IGF receptor is stimulated. In addition, literally all of the regulatory proteins have suspected or known activities that are not directly related to IGF signaling. How such activities may integrate with IGF signaling is also important to address in the future.
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Factor I del Crecimiento Similar a la Insulina , Proteína Plasmática A Asociada al Embarazo , Animales , Humanos , Proteína Plasmática A Asociada al Embarazo/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Glicoproteínas/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Receptores de Somatomedina/metabolismo , Proteína 4 de Unión a Factor de Crecimiento Similar a la InsulinaRESUMEN
The metzincin metalloproteinase PAPP-A plays a key role in the regulation of insulin-like growth factor (IGF) signaling by specific cleavage of inhibitory IGF binding proteins (IGFBPs). Using single-particle cryo-electron microscopy (cryo-EM), we here report the structure of PAPP-A in complex with its endogenous inhibitor, stanniocalcin-2 (STC2), neither of which have been reported before. The highest resolution (3.1 Å) was obtained for the STC2 subunit and the N-terminal approximately 1000 residues of the PAPP-A subunit. The 500 kDa 2:2 PAPP-A·STC2 complex is a flexible multidomain ensemble with numerous interdomain contacts. In particular, a specific disulfide bond between the subunits of STC2 and PAPP-A prevents dissociation, and interactions between STC2 and a module located in the very C-terminal end of the PAPP-A subunit prevent binding of its main substrate, IGFBP-4. While devoid of activity towards IGFBP-4, the active site cleft of the catalytic domain is accessible in the inhibited PAPP-A·STC2 complex, as shown by its ability to hydrolyze a synthetic peptide derived from IGFBP-4. Relevant to multiple human pathologies, this unusual mechanism of proteolytic inhibition may support the development of specific pharmaceutical agents, by which IGF signaling can be indirectly modulated.
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Hormonas Peptídicas , Somatomedinas , Humanos , Microscopía por Crioelectrón , Disulfuros/metabolismo , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Hormonas Peptídicas/metabolismo , Péptido Hidrolasas/metabolismo , Proteína Plasmática A Asociada al Embarazo/química , Proteína Plasmática A Asociada al Embarazo/metabolismo , Somatomedinas/metabolismoRESUMEN
CONTEXT: Pappalysins (PAPP-A, PAPP-A2) modulate body growth by increasing insulin-like growth factor I (IGF-I) bioavailability through cleavage of insulin-like growth factor binding proteins (IGFBPs) and are inhibited by stanniocalcins (STC1, STC2). Normative data on these novel factors, as well as on free IGF-I and uncleaved fractions of IGFBPs, are not well established. OBJECTIVE: This work aimed to determine serum concentrations of PAPP-A, PAPP-A2, STC1, and STC2 in relationship with other growth hormone (GH)-IGF axis parameters during development. METHODS: Full-term newborns (150; gestational age: 39.30â ±â 1.10 weeks), 40 preterm newborns (30.87â ±â 3.35 weeks), and 1071 healthy individuals (aged 1-30 years) were included in the study and divided according to their Tanner stages (males and females): I:163 males, 154 females; II:100 males, 75 females; III:83 males, 96 females; IV: 77 males, 86 females; and V:109 males,128 females. RESULTS: Serum concentrations of PAPP-A, PAPP-A2, STC1, STC2, IGFBP-2, total IGFBP-4, and total IGFBP-5 were elevated at birth and declined throughout childhood. In postnatal life, PAPP-A2 concentrations decreased progressively in concomitance with the free/total IGF-I ratio; however, stanniocalcin concentrations remained stable. PAPP-A2 concentrations positively correlated with the free/total IGF-I ratio (râ =â +0.28; Pâ <â .001) and negatively with the intact/total IGFBP-3 ratio (râ =â -0.23; Pâ <â .001). PAPP-A concentrations inversely correlated with intact/total IGFBP-4 ratio (râ =â -0.21; Pâ <â .001), with PAPP-A concentrations being lower in females at all ages. Association studies indicate the importance of stanniocalcins and pappalysins in the control of this axis in an age-specific manner. CONCLUSION: This study provides reference values of pappalysins and stanniocalcins, which modulate IGF-I activity by changing the concentrations of cleaved and uncleaved IGFBPs.
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Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina , Factor I del Crecimiento Similar a la Insulina , Niño , Femenino , Glicoproteínas , Hormona del Crecimiento/metabolismo , Humanos , Lactante , Recién Nacido , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Proteína Plasmática A Asociada al Embarazo/metabolismoRESUMEN
BACKGROUND: Diabetic nephropathy (DN) is a serious complication of diabetes and a common cause of end stage renal failure. Insulin-like growth factor (IGF)-signaling has been implicated in DN, but is mechanistically poorly understood. Here, we assessed the activity of the metalloproteinase PAPP-A, an activator of IGF activity, and its possible interaction with the endogenous PAPP-A inhibitors stanniocalcin (STC)-1 and -2 in the mammalian kidney under normal and hyperglycemic conditions. METHODS AND RESULTS: Immunohistochemistry demonstrated that PAPP-A, its proteolytic substrate IGF binding protein-4, STC1 and STC2 are present in the human kidney. Endogenous inhibited complexes of PAPP-A (PAPP-A:STC1 and PAPP-A:STC2) were demonstrated in media conditioned by human mesangial cells (HMCs), suggesting that PAPP-A activity is regulated by the STCs in kidney tissue. A method for the selective detection of active PAPP-A in tissue was developed and a significant increase in glomerular active PAPP-A in human diabetic kidney relative to normal was observed. In DN patients, the estimated glomerular filtration rate correlated with PAPP-A activity. In diabetic mice, glomerular growth was reduced when PAPP-A activity was antagonized by adeno-associated virus-mediated overexpression of STC2. CONCLUSION: We propose that PAPP-A activity in renal tissue is precisely balanced by STC1 and STC2. An imbalance in this equilibrium causing increased PAPP-A enzymatic activity potentially contributes to the development of DN, and thus, therapeutic targeting of PAPP-A activity may represent a novel strategy for its treatment.
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Diabetes Mellitus Experimental , Nefropatías Diabéticas , Proteína Plasmática A Asociada al Embarazo , Animales , Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/etiología , Humanos , Hipertrofia , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mamíferos/metabolismo , Ratones , Proteína Plasmática A Asociada al Embarazo/metabolismo , ProteolisisRESUMEN
Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer, and it exhibits a number of clinico-pathological characteristics distinct from the more common invasive ductal carcinoma (IDC). We set out to identify alterations in the tumor microenvironment (TME) of ILC. We used laser-capture microdissection to separate tumor epithelium from stroma in 23 ER+ ILC primary tumors. Gene expression analysis identified 45 genes involved in regulation of the extracellular matrix (ECM) that were enriched in the non-immune stroma of ILC, but not in non-immune stroma from ER+ IDC or normal breast. Of these, 10 were expressed in cancer-associated fibroblasts (CAFs) and were increased in ILC compared to IDC in bulk gene expression datasets, with PAPPA and TIMP2 being associated with better survival in ILC but not IDC. PAPPA, a gene involved in IGF-1 signaling, was the most enriched in the stroma compared to the tumor epithelial compartment in ILC. Analysis of PAPPA- and IGF1-associated genes identified a paracrine signaling pathway, and active PAPP-A was shown to be secreted from primary CAFs. This is the first study to demonstrate molecular differences in the TME between ILC and IDC identifying differences in matrix organization and growth factor signaling pathways.
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Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with few effective treatment options. We found a highly significant correlation between pregnancy-associated plasma protein (PAPP)-A expression in IPF lung tissue and disease severity as measured by various pulmonary and physical function tests. PAPP-A is a metalloproteinase that enhances local insulin-like growth factor (IGF) activity. We used primary cultures of normal adult human lung fibroblasts (NHLF) to test the hypothesis that PAPP-A plays an important role in the development of pulmonary fibrosis. Treatment of NHLF with pro-fibrotic transforming growth factor (TGF)-ß stimulated marked increases in IGF-I mRNA expression (>20-fold) and measurable IGF-I levels in 72-h conditioned medium (CM). TGF-ß treatment also increased PAPP-A levels in CM fourfold (p = 0.004) and proteolytic activity ~2-fold. There was an indirect effect of TGF-ß to stimulate signaling through the PI3K/Akt pathway, which was significantly inhibited by both IGF-I-inactivating and PAPP-A inhibitory antibodies. Induction of senescence in NHLF increased PAPP-A levels in CM 10-fold (p = 0.006) with attendant increased proteolytic activity. Thus, PAPP-A is a novel component of the senescent lung fibroblast secretome. In addition, NHLF secreted extracellular vehicles (EVs) with surface-bound active PAPP-A that were increased fivefold with senescence. Regulation of PAPP-A and IGF signaling by TGF-ß and cell senescence suggests an interactive cellular mechanism underlying the resistance to apoptosis and the progression of fibrosis in IPF. Furthermore, PAPP-A-associated EVs may be a means of pro-fibrotic, pro-senescent communication with other cells in the lung and, thus, a potential therapeutic target for IPF.
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Fibrosis Pulmonar Idiopática , Proteína Plasmática A Asociada al Embarazo/metabolismo , Adulto , Medios de Cultivo Condicionados/farmacología , Fibroblastos/metabolismo , Fibrosis , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Plasmática A Asociada al Embarazo/genética , Proteína Plasmática A Asociada al Embarazo/farmacología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Pregnancy-associated plasma protein-A (PAPP-A) is an enzyme that increases IGF-activity through cleavage of IGF-binding proteins (IGFBPs), primarily IGFBP-4, whereby bound IGF-I becomes released as a free molecule. The enzymatic activity of PAPP-A is irreversibly suppressed by the glycoprotein stanniocalcin-2 (STC2). Pre-clinical and clinical studies suggest that the STC2 - PAPP-A - IGFBP-4 axis is important in controlling local IGF-action. STC2, PAPP-A and IGFBP-4 are expressed in adipose tissue, and as bariatric surgery markedly reduces the amount of fat, we found it relevant to study the impact of Roux-en-Y gastric bypass (RYGB) on circulating concentrations of this IGF-regulatory network. METHODS: Analysis of fasting blood samples from 20 obese subjects, hereof 10 with preoperative type 2 diabetes, investigated before RYGB, and 1â¯week, 3â¯months and 12â¯months post-surgery. Members of the IGF-system were analyzed by immunoassays, bioactive IGF by cell-based IGF-I receptor activation assay. We compared changes in IGF-system components with changes in fasting plasma insulin and glucose, and HbA1c. RESULTS: PAPP-A remained unchanged, but STC2 decreased following RYGB (pâ¯<â¯0.05). The PAPP-A substrate IGFBP-4 declined (pâ¯<â¯0.01), whereas levels of PAPP-A specific IGFBP-4 fragments increased (pâ¯<â¯0.05), indicating an increased PAPP-A enzymatic activity post-RYGB. Further, the reduction in intact IGFBP-4 correlated with increased levels of bioactive IGF (pâ¯<â¯0.05). In multivariable regression analyses, an improved glucose metabolism correlated with reductions in STC2 and IGFBP-4, and with increases in bioactive IGF and IGF-I (pâ¯<â¯0.05). CONCLUSION: After 12â¯months, RYGB caused reduced serum concentrations of intact IGFBP-4 and STC2, whereas serum PAPP-A remained at pre-operative levels. However, concentrations of PAPP-A generated IGFBP-4 fragments increased, pointing to an overall increased PAPP-A enzymatic activity following RYGB. Notably, reductions in intact IGFBP-4 and STC2 associated with improvements in glucose metabolism. Therefore, we propose that STC2 and IGFBP-4 are involved in the metabolic improvement that follows RYGB.
Asunto(s)
Derivación Gástrica , Glicoproteínas/sangre , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Factor I del Crecimiento Similar a la Insulina/análisis , Péptidos y Proteínas de Señalización Intercelular/sangre , Obesidad Mórbida/cirugía , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Obesidad Mórbida/sangre , Proteína Plasmática A Asociada al Embarazo/análisisRESUMEN
Superoxide dismutase 3 (SOD3) is an extracellular protein with the capacity to convert superoxide into hydrogen peroxide, an important secondary messenger in redox regulation. To investigate the utility of zebrafish in functional studies of SOD3 and its relevance for redox regulation, we have characterized the zebrafish orthologues; Sod3a and Sod3b. Our analyses show that both recombinant Sod3a and Sod3b express SOD activity, however, only Sod3b is able to bind heparin. Furthermore, RT-PCR analyses reveal that sod3a and sod3b are expressed in zebrafish embryos and are present primarily in separate organs in adult zebrafish, suggesting distinct functions in vivo. Surprisingly, both RT-PCR and whole mount in situ hybridization showed specific expression of sod3b in skeletal tissue. To further investigate this observation, we compared femoral bone obtained from wild-type and SOD3-/- mice to determine whether a functional difference was apparent in healthy adult mice. Here we report, that bone from SOD3-/- mice is less mineralized and characterized by significant reduction of cortical and trabecular thickness in addition to reduced mechanical strength. These analyses show that SOD3 plays a hitherto unappreciated role in bone development and homeostasis.