Fibrillins in Adult Human Ovary and Polycystic Ovary Syndrome: Is Fibrillin-3 Affected in PCOS?

Polycystic ovary syndrome (PCOS) is a common endocrinopathy in women of reproductive age. Although genetic linkage analyses have demonstrated a susceptibility locus for PCOS mapping to the fibrillin-3 gene, the presence of fibrillin proteins in normal and polycystic ovaries has not been characterized. This study compared and contrasted fibrillin-1, -2, and -3 localization in normal and polycystic ovaries. Immunohistochemical stainings of ovaries from 21 controls and 9 patients with PCOS were performed. Fibrillin-1 was ubiquitous in ovarian connective tissue. Fibrillin-2 localized around antral follicles and in areas of folliculolysis. Fibrillin-3 was present in a restricted distribution within the specialized perifollicular stroma of follicles in morphological transition from primordial to primary type [transitional follicles (TFs)]. Fibrillin-1 and -2 stainings of PCOS ovaries were similar to those of the controls. However, in eight of the nine PCOS ovaries, there was a decrease in the number of TFs associated with fibrillin-3, including no staining in five PCOS samples; decreased number of fibrillin-3-associated TFs/mm2 was confirmed by quantitative analysis. Our findings support a role for fibrillin-3 in the pathogenesis of PCOS and suggest fibrillin-3 may function in primordial to primary follicle transition. We propose that loss of fibrillin-3 during folliculogenesis may be an important factor in PCOS pathogenesis. (J Histochem Cytochem 58:903–915, 2010

Microenvironmental Regulation by Fibrillin-1

Fibrillin-1 is a ubiquitous extracellular matrix molecule that sequesters latent growth factor complexes. A role for fibrillin-1 in specifying tissue microenvironments has not been elucidated, even though the concept that fibrillin-1 provides extracellular control of growth factor signaling is currently appreciated. Mutations in FBN1 are mainly responsible for the Marfan syndrome (MFS), recognized by its pleiotropic clinical features including tall stature and arachnodactyly, aortic dilatation and dissection, and ectopia lentis. Each of the many different mutations in FBN1 known to cause MFS must lead to similar clinical features through common mechanisms, proceeding principally through the activation of TGFβ signaling. Here we show that a novel FBN1 mutation in a family with Weill-Marchesani syndrome (WMS) causes thick skin, short stature, and brachydactyly when replicated in mice. WMS mice confirm that this mutation does not cause MFS. The mutation deletes three domains in fibrillin-1, abolishing a binding site utilized by ADAMTSLIKE-2, -3, -6, and papilin. Our results place these ADAMTSLIKE proteins in a molecular pathway involving fibrillin-1 and ADAMTS-10. Investigations of microfibril ultrastructure in WMS humans and mice demonstrate that modulation of the fibrillin microfibril scaffold can influence local tissue microenvironments and link fibrillin-1 function to skin homeostasis and the regulation of dermal collagen production. Hence, pathogenetic mechanisms caused by dysregulated WMS microenvironments diverge from Marfan pathogenetic mechanisms, which lead to broad activation of TGFβ signaling in multiple tissues. We conclude that local tissue-specific microenvironments, affected in WMS, are maintained by a fibrillin-1 microfibril scaffold, modulated by ADAMTSLIKE proteins in concert with ADAMTS enzymes.Sengle G, Tsutsui K, Keene DR, Tufa SF, Carlson EJ, Charbonneau NL, et al. (2012) Microenvironmental Regulation by Fibrillin-1. PLoS Genet 8(1): e1002425. https://doi.org/10.1371/journal.pgen.100242

Abnormal Activation of BMP Signaling Causes Myopathy in <i>Fbn2</i> Null Mice

<div><p>Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that <i>Fbn2</i> null mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in <i>Fbn2</i> null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult <i>Fbn2</i> null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult <i>Fbn2</i> null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, <i>Fbn2</i> null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that <i>FBN2</i> should be considered as a candidate gene for recessive congenital muscular dystrophy. Both <i>in vivo</i> and <i>in vitro</i> evidence associated muscle abnormalities and accumulation of white fat in <i>Fbn2</i> null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in <i>Fbn2</i> null mice was accomplished by deleting a single allele of <i>Bmp7</i>. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.</p></div

Muscle architecture and forelimb fat at P8.

<p><b>(A)</b> H&E stained cross sections of wildtype and <i>Fbn2</i> null forearms at P8 <b>(left)</b> and quantitation of percentages of myofibers with central nuclei <b>(right)</b>. Quantitation was performed as in 1C and 1D. <b>(B)</b> Toluidine Blue stained cross sections of P8 wildtype and <i>Fbn2</i> null forearms showing accumulation of fat droplets (filled grey spheres) in the null forearm. Bars = 50 μm in <b>(A)</b> and <b>(B)</b>. <b>(C)</b> Transmission electron microscopy of fat droplets in P8 <i>Fbn2</i> null forearm muscle. The micrograph was taken from a region containing fat droplets in the block shown in (<b>B</b>). Bar = 10 μm. <b>(D)</b> A comparable series of 3 μm thick sections of wildtype and <i>Fbn2</i> null forearms generated by micro-CT (sections numbered above each panel). Fat and bone appear white; fat is solid white, while bone includes grey space within the circular bone collar. Bars = 1 mm. <b>(E)</b> qPCR anaysis of fat specific markers in <i>Fbn2</i> heterozygous and null mice compared to wildtype littermates.</p

Primers used for quantitative real-time PCR.

<p>Primers used for quantitative real-time PCR.</p

In vitro BMP bioactivity assay using C2C12 cells.

<p><b>(A)</b> Comparison of <i>Fbn1</i> and <i>Fbn2</i> mRNA levels during myoblast to myotube differentiation. <b>(B)</b> BMP activity measured with and without RNAi of <i>Fbn2</i> during myoblast to myotube differentiation. <b>(C)</b> Domain structure of fibrillin-2 and recombinant polypeptide rF86. <b>(D)</b> Interaction of fibrillin-2 with the prodomain of BMP-7 complex confers latency to the growth factor. <b>Top left:</b> cartoon depicting BMP-7 complex, a growth factor dimer non-covalently associated with two processed prodomains. <b>Top right:</b> cartoon of the assay: BMP-7 complex was coupled via prodomain interactions either through a mAb directed against the N-terminal His₆-tag of the BMP-7 prodomain or an N-terminal fibrillin-2 peptide (rF86) which was captured by mAb689. C2C12 cells were then seeded. <b>Below:</b> quantitation of coupled amounts of immobilized BMP-7 complex determined by comparison with standards of known concentration. <b>(E)</b> qPCR expression levels of <i>Id3</i> from treated and untreated wells. Error bars indicate mean ± SD. Each experiment was performed in triplicates.</p

Primers for generating template standards to absolutely quantitate BMP mRNA amounts.

<p>Primers for generating template standards to absolutely quantitate BMP mRNA amounts.</p

BMP signaling in wildtype and <i>Fbn2</i> null forearm muscle.

<p><b>(A)</b> Phosphosmad 1/5/8 immunofluorescence signals (green dots) within DAPI blue-stained nuclei of <i>Fbn2</i> null muscle compared to wildtype at P1 and P8. <b>(B)</b><i>BMP</i> mRNA expression levels in P0 <i>Fbn2</i> heterozygous and null muscle compared to wildtype. <b>(C)</b> Absolute expression levels of <i>Bmp</i> mRNA in P0 wildtype forearm muscles. N = the number of animals analyzed. Error bars indicate mean ± SD. Bars = 50 μm.</p