Serine Protease HTRA1 Antagonizes Transforming Growth Factor-β Signaling by Cleaving Its Receptors and Loss of HTRA1 <i>In Vivo</i> Enhances Bone Formation

<div><p>HTRA1 is a member of the High Temperature Requirement (HTRA1) family of serine proteases, which play a role in several biological and pathological processes. In part, HTRA1 regulation occurs by inhibiting the TGF-β signaling pathway, however the mechanism of inhibition has not been fully defined. Previous studies have shown that HTRA1 is expressed in a variety of tissues, including sites of skeletal development. HTRA1 has also been implicated in the process of bone formation, although the precise manner of regulation is still unknown. This study investigated how HTRA1 regulates TGF-β signaling and examined the <i>in vivo</i> effects of the loss of HTRA1. We demonstrated that recombinant HTRA1 was capable of cleaving both type II and type III TGF-β receptors (TβRII and TβRIII) <i>in vitro</i> in a dose-dependent manner, but it did not affect the integrity of TβRI or TGF-β. Overexpression of HTRA1 led to decreased levels of both TβRII and III on the cell surface but had no effect on TβRI. Silencing HTRA1 expression significantly increased TGF-β binding to the cell surface and TGF-β responsiveness within the cell. To examine the role of HTRA1 <i>in vivo</i>, we generated mice with a targeted gene deletion of <i>HTRA1</i>. Embryonic fibroblasts isolated from these mice displayed an increase in TGF-β-induced expression of several genes known to promote bone formation. Importantly, the loss of HTRA1 in the knockout mice resulted in a marked increase in trabecular bone mass. This study has identified a novel regulatory mechanism by which HTRA1 antagonizes TGF-β signaling, and has shown that HTRA1 plays a key role in the regulation of bone formation.</p></div

HTRA1 cleaves TGF-β receptors.

<p>(<i>A</i>) Cleavage of TGF-β receptors by HTRA1 <i>in vitro</i>. Equal amounts of purified TGF-β receptors were incubated with increasing amount of purified HTRA1 with or without CPII, an HTRA1 agonist. The inactive mutant, S328A, was also incubated with the receptors in the presence or absence of CPII. Protein digestions were analyzed by western blot. UD = undigested proteins. (<i>B</i>) HTRA1 cleavage of TGF-β receptors from the cell surface. HeLa cells were transfected with either a control or HTRA1 plasmid in addition to plasmids encoding either TβRIII, TβRII or TβRI. Membrane proteins were isolated and analyzed by western blot for cleavage of membrane bound receptors. Cadherin was used as a loading control. Overexpression of His-tagged HTRA1 was confirmed with an anti-His antibody. (<i>C</i>) mRNA levels of the TGF-β receptors after HeLa cells were transfected with either a control or HTRA1 plasmid.</p

Regulation of TGF-β signaling by HTRA1.

<p>(<i>A</i>)(<i>B</i>) Binding of TGF-β to the cell surface as measured by flow cytometry. Cells were transfected with either nonspecific control siRNA or HTRA1 siRNA and then incubated with either biotinylated TGF-β or a biotinylated negative control protein to measure the background. Solid gray = background, dashed line = control siRNA, black line = HTRA1 siRNA. (<i>B</i>) Bar chart indicates the mean FITC values after subtraction of the background level. **<i>P</i><0.01, Student’s <i>t</i>-test, n = 4. (<i>C</i>) Immunoblot of total and phosphorylated Smad2 (465/467) in A549 cells transfected with either control or HTRA1 siRNA, and then treated with 1 ng/ml TGF-β for the indicated times. β-actin was used as a loading control. (<i>D</i>) The effect of HTRA1 knockdown on the expression of TGF-β-regulated genes. A549 cells were transfected with either nonspecific control siRNA or HTRA1 siRNA and then treated with 1 ng/ml TGF-β for the indicated times. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, Student’s <i>t</i>-test, n = 3.</p

The <i>in vivo</i> effects of loss of HTRA1.

<p>(<i>A</i>) Schematic of the <i>HTRA1</i> knockout strategy. Primers were designed with one forward primer (For) that amplifies an amplicon for the wild type and heterozygous alleles with a corresponding reverse primer (Rev), and a second reverse primer designed to only amplify the knockout. The knockout reverse primer (KO Rev) only amplifies following CRE-LOX recombination. Solid triangles = loxP sites; EV = EcoRI. E2 = Exon2; E3 = Exon3. (<i>B</i>) Genotype results of mouse embryonic fibroblasts showing confirmation of the generation of <i>HTRA1</i>+/− and <i>HTRA1</i>−/− mice. (<i>C</i>) Expression of TGF-β-regulated genes in embryonic fibroblasts from wild type (WT) and <i>HTRA1</i> knockout (KO) mice. Cells were treated with 1 ng/ml TGF-β for the indicated times. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, Student’s <i>t</i>-test, n = 9 for each cell type. (<i>D</i>) Microcomputed tomography and parameters of trabecular bone in the distal femurs in wild type (WT), <i>HTRA1</i> heterozygous (HET) and <i>HTRA1</i> knockout (KO) mice (all males, 3 months old). (<i>E</i>) Microcomputed tomography and parameters of trabecular bone in the fourth vertebrae in wild type (WT), <i>HTRA1</i> heterozygous (HET) and <i>HTRA1</i> knockout (KO) mice. For both (<i>D</i>) and (<i>E</i>), *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ANOVA and Fisher’s protected least significant difference test, n = 13.</p

HTRA1 inhibits TGF-β-mediated transcription.

<p>(<i>A</i>) The effect of HTRA1 on TGF-β transcription was examined by co-transfecting HeLa cells with a luciferase reporter plasmid driven by tandem Smad responsive elements and with either control plasmid or 0, 25, 50, 100 or 150 ng of a plasmid that overexpresses HTRA1. After 24 hours, cells were either left untreated or treated with 10 ng/ml TGF-β for 16 hours to induce the Smad-responsive reporter gene. Relative luciferase activity was determined by calculating the ratio of firefly and <i>Renilla</i> luciferase activities. (<i>B</i>) The effect of exogenous treatment of HTRA1 on TGF-β-mediated transcription. HeLa cells were transfected with a Smad-responsive reporter and then either left untreated or treated with the indicated concentrations of purified HTRA1 or an inactive mutant, S328A. Relative luciferase activity was determined by calculating the ratio of firefly and <i>Renilla</i> luciferase activities. (<i>C</i>) TGF-β-induced expression of <i>PAI-1</i> mRNA. HeLa cells were transfected with either a control plasmid or an HTRA1 plasmid, and then treated with 10 ng/ml of TGF-β for the indicated times. (<i>D</i>) Expression of <i>PAI-1</i> mRNA in HeLa cells transfected with either a nonspecific control siRNA or with HTRA1 siRNA, then treated with 10 ng/ml of TGF-β for the indicated times. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, compared to TGF-β treated control (<i>A</i>, <i>B</i>), or empty plasmid and non-specific siRNA controls (<i>C</i>, <i>D</i>). Student’s <i>t</i>-test, n = 3.</p

Maestros que hacen historia / tejedores de sentidos : entre voces, silencios y memorias

La intención del presente proyecto-libro es generar procesos colectivos que permitan reflexionar, a buscar formas de nombrar metodológica y epistemáticamente los trazos de las acciones de las memorias en movimiento, cuyos puntos de articulación son los territorios -tejidos y huecos- de los silencios, de los olvidos; de la emergencia de las narrativas que transitan por otras formas y modos de hacer historia a través de la oralidad, en sus múltiples temporalidades y rostros. Por tanto, Maestros que hacen historia, reúne a docentes-investigadores que desarrollan la práctica creativa, tanto que la formación pedagógica a través de la tensión reflexiva sobre los relatos/testimonios, como de la creación de colectivos pedagógicos, en particular sobre historia oral en nuestro continente