Extravillous trophoblast-associated ADAM12 exerts pro-invasive properties, including induction of integrin beta 1-mediated cellular spreading.

ADAM12, consisting of a membrane-bound (ADAM12L) and a secreted (ADAM12S) form, is expressed exclusively in regenerating and developing tissue as well as in certain cancer types. Strong ADAM12 expression levels have been noticed in the human placenta, and deregulated ADAM12S levels were associated with various pregnancy-related disorders including pre-eclampsia and intrauterine growth restriction. However, the role of ADAM12 in trophoblast motility has not been investigated so far. Hence, the present study aimed to investigate the specific function of the protease by using different primary trophoblast cell models. Immunofluorescence and Western blot analyses of first trimester placental tissue and differentiating primary first trimester cytotrophoblasts (CTBs) indicated strong upregulation of both of the ADAM12 isoforms during extravillous trophoblast differentiation. Functional assays involving short interfering RNA (siRNA)-mediated knockdown studies in primary CTBs and first trimester explant cultures revealed a significant repression of trophoblast motility upon partial loss of ADAM12. Conversely, isoform-specific overexpression in the ADAM12-negative trophoblast cell line SGHPL-5 enhanced the invasive capacity of these cells. We further confirmed proteolytic activity of trophoblast-derived ADAM12S by demonstrating its potential to degrade insulin-like growth factor-binding protein 3. Finally, we suggest that ADAM12S exerts its pro-migratory function in trophoblasts by inducing integrin beta 1-mediated cellular spreading.

The muscle dystrophy-causing ΔK32 lamin A/C mutant does not impair the functions of the nucleoplasmic lamin-A/C-LAP2α complex in mice.

A-type lamins are components of the nuclear lamina, a filamentous network of the nuclear envelope in metazoans that supports nuclear architecture. In addition, lamin A/C can also be found in the interior of the nucleus. This nucleoplasmic lamin pool is soluble in physiological buffer, depends on the presence of the lamin-binding protein, lamina-associated polypeptide 2α (LAP2α) and regulates cell cycle progression in tissue progenitor cells. ΔK32 mutations in A-type lamins cause severe congenital muscle disease in humans and a muscle maturation defect in Lmna(ΔK32/ΔK32) knock-in mice. Mutant ΔK32 lamin A/C protein levels were reduced and all mutant lamin A/C was soluble and mislocalized to the nucleoplasm. To test the role of LAP2α in nucleoplasmic ΔK32 lamin A/C regulation and functions, we deleted LAP2α in Lmna(ΔK32/ΔK32) knock-in mice. In double mutant mice the Lmna(ΔK32/ΔK32)-linked muscle defect was unaffected. LAP2α interacted with mutant lamin A/C, but unlike wild-type lamin A/C, the intranuclear localization of ΔK32 lamin A/C was not affected by loss of LAP2α. In contrast, loss of LAP2α in Lmna(ΔK32/ΔK32) mice impaired the regulation of tissue progenitor cells as in lamin A/C wild-type animals. These data indicate that a LAP2α-independent assembly defect of ΔK32 lamin A/C is the predominant cause of the mouse pathology, whereas the LAP2α-linked functions of nucleoplasmic lamin A/C in the regulation of tissue progenitor cells are not affected in Lmna(ΔK32/ΔK32) mice.

Endostatin suppresses IGF-II-mediated signaling and invasion of human extravillous trophoblasts.

Endostatin, a biological active fragment of the extracellular matrix protein collagen XVIII, is known to interfere with cellular motility in the context of pathological angiogenesis. However, the physiological role of endostatin remains largely elusive. Recent evidence suggested that the inhibitor is produced in human decidual cells of early pregnancy, indicating that endostatin could be involved in diverse reproductive processes, such as implantation and/or placental differentiation. To gain more insights into the role of endostatin, we here analyzed its effects on trophoblast motility, proliferation, and signaling using purified primary trophoblasts, first-trimester villous explant cultures, and trophoblastic SGHPL-5 cells. In vitro Transwell assays demonstrated that purified endostatin inhibited both basal and IGF-II-induced migration and invasion as well as outgrowth from villous explant cultures. In contrast, basal and IGF-II-stimulated proliferation was unaffected upon addition of the inhibitor. Analyses of IGF-II-associated downstream signaling events showed that endostatin interfered with activation of various signaling kinases such as ERK1/2, protein kinase B (Akt)/mammalian target of rapamycin/p70 S6 kinase, and focal adhesion kinase. Furthermore, virus-mediated, stable gene silencing of Akt1 in SGHPL-5 cells using a micro-RNA-adapted short hairpin RNA-expressing plasmid revealed that endostatin-mediated inhibition of IGF-II-induced Akt phosphorylation was critically dependent on the expression of the particular isoform. In conclusion, the data suggest that endostatin could be a physiological inhibitor of IGF-II-dependent trophoblast cell motility by suppressing focal adhesion kinase/Akt/mammalian target of rapamycin/p70 S6 kinase signaling.

Transcription factor AP-2α promotes EGF-dependent invasion of human trophoblast.

The basic helix-span-helix transcription factor activating protein (AP)-2α is critically involved in cell-specific hormone expression of syncytializing human trophoblasts. Its role in invasive trophoblast differentiation, however, remains largely elusive. Using RT-PCR, Western blotting, and immunofluorescence of first-trimester placentae, we here show that AP-2α is expressed in extravillous trophoblasts (EVTs) both in situ and in vitro as well as in invasive trophoblast cell lines. Its protein expression was increased upon supplementation of epidermal growth factor (EGF) both in primary EVTs and trophoblastic SGHPL-5 cells. Gene silencing of AP-2α using small hairpin microRNA (shRNAmir) did not affect basal invasion of SGHPL-5 cells through Matrigel-coated filters but reduced EGF-stimulated invasion. Similarly, treatment of primary EVTs with AP-2α small interfering RNA decreased EGF-dependent invasion. Proliferation of SGHPL-5 cells and primary EVTs, measured by cumulative cell numbers and 5-bromo-2'-deoxyuridine labeling, respectively, were not affected on loss of AP-2α. EGF-dependent induction of matrix metalloproteinase (MMP)-2, pro- and active form of urokinase plasminogen activator, and chorionic gonadotropin (CG)-ß was noticed in shRNAmir-control cells, whereas these genes were suppressed in EGF-treated shRNAmir-AP-2α cells. Similarly, EGF-stimulated MMP-2 and CGß protein expression was reduced in AP-2α small interfering RNA-treated primary EVTs. Knockdown of AP-2α also decreased luciferase activity of the CGß5 promoter in SGHPL-5 cells, which was compensated upon transient overexpression of AP-2α cDNA. In conclusion, we show that AP-2α expression positively affects human trophoblast invasion under EGF-stimulated conditions, likely by inducing critical invasion-promoting genes such MMP-2, urokinase plasminogen activator, and CG.

Lamina-associated polypeptide 2alpha loss impairs heart function and stress response in mice.

RATIONALE: Lamina-associated polypeptide (LAP)2alpha is a mammalian chromatin-binding protein that interacts with a fraction of A-type lamins in the nuclear interior. Because mutations in lamins and LAP2alpha lead to cardiac disorders in humans, we hypothesized that these factors may play important roles in heart development and adult tissue homeostasis. OBJECTIVE: We asked whether the presence of LAP2alpha was required for normal cardiac function. METHODS AND RESULTS: To study the molecular mechanisms of the disease, we analyzed heart structure and function in complete and conditional Lap2alpha(-/-) mice as well as Lap2alpha(-/-)/Mdx mutants. Unlike conditional deletion of LAP2alpha in late embryonic striated muscle, its complete knockout caused systolic dysfunction in young mice, accompanied by sporadic fibrosis in old animals, as well as deregulation of major cardiac transcription factors GATA4 and myocyte enhancer factor 2c. Activation of compensatory pathways, including downregulation of beta-adrenergic receptor signaling, resulted in reduced responsiveness of the myocardium to chronic beta-adrenergic stimulation and stalled the progression of LAP2alpha-deficient hearts from hypertrophy toward cardiac failure. Dystrophin deficiency in an Mdx background resulted in a transient rescue of the Lap2alpha(-/-) phenotype. CONCLUSIONS: Our data suggest a novel role of LAP2alpha in the maintenance of cardiac function under normal and stress conditions.

Loss of LAP2 alpha delays satellite cell differentiation and affects postnatal fiber-type determination.

Lamina-associated polypeptide 2 alpha (LAP2 alpha) is a nucleoplasmic protein implicated in cell cycle regulation through its interaction with A-type lamins and the retinoblastoma protein. Mutations in lamin A/C and LAP2 alpha cause late onset striated muscle diseases, but the molecular mechanisms are poorly understood. To study the role of LAP2 alpha in skeletal muscle function and postnatal tissue homeostasis, we generated complete and muscle-specific LAP2 alpha knockout mice. Whereas overall muscle morphology, function, and regeneration were not detectably affected, the myofiber-associated muscle stem cell pool was increased in complete LAP2 alpha knockout animals. At molecular level, the absence of LAP2 alpha preserved the stem cell-like phenotype of Lap2 alpha(-/-) primary myoblasts and delayed their in vitro differentiation. In addition, loss of LAP2 alpha shifted the myofiber-type ratios of adult slow muscles toward fast fiber types. Conditional Cre-mediated late muscle-specific ablation of LAP2 alpha affected early stages of in vitro myoblast differentiation, and also fiber-type determination, but did not change myofiber-associated stem cell numbers in vivo. Our data demonstrate multiple and distinct functions of LAP2 alpha in muscle stem cell maintenance, early phases of myogenic differentiation, and muscle remodeling.