Tyrosine Sulfation of Native Mouse Psgl-1 Is Required for Optimal Leukocyte Rolling on P-Selectin In Vivo

We recently demonstrated that tyrosine sulfation is an important contributor to monocyte recruitment and retention in a mouse model of atherosclerosis. P-selectin glycoprotein ligand-1 (Psgl-1) is tyrosine-sulfated in mouse monocyte/macrophages and its interaction with P-selectin is important in monocyte recruitment in atherosclerosis. However, whether tyrosine sulfation is required for the P-selectin binding function of mouse Psgl-1 is unknown. Here we test the function of native Psgl-1 expressed in leukocytes lacking endogenous tyrosylprotein sulfotransferase (TPST) activity.Psgl-1 function was assessed by examining P-selectin dependent leukocyte rolling in post-capillary venules of C57BL6 mice transplanted with hematopoietic progenitors from wild type (WT → B6) or Tpst1;Tpst2 double knockout mice (Tpst DKO → B6) which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules. Similar results were observed on immobilized P-selectin in vitro. Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.These findings provide direct and convincing evidence that tyrosine sulfation is required for optimal function of mouse Psgl-1 in vivo and suggests that tyrosine sulfation of Psgl-1 contributes to the development of atherosclerosis

MiR-126 and miR-126* regulate shear-resistant firm leukocyte adhesion to human brain endothelium

Leukocyte adhesion to brain endothelial cells, the blood-brain barrier main component, is a critical step in the pathogenesis of neuroinflammatory diseases such as multiple sclerosis (MS). Leukocyte adhesion is mediated mainly by selectins, cell adhesion molecules and chemokines induced by pro-inflammatory cytokines such as TNFα and IFNγ, but the regulation of this process is not fully clear. This study investigated the regulation of firm leukocyte adhesion to human brain endothelium by two different brain endothelial microRNAs (miRs), miR-126 and miR-126*, that are downregulated by TNFα and IFNγ in a human brain endothelial cell line, hCMEC/D3. Using a leukocyte adhesion in vitro assay under shear forces mimicking blood flow, we observed that reduction of endothelial miR-126 and miR-126* enhanced firm monocyte and T cell adhesion to hCMEC/D3 cells, whereas their increased expression partially prevented THP1, Jurkat and primary MS patient-derived PBMC firm adhesion. Furthermore, we observed that miR-126* and miR-126 downregulation increased E-selectin and VCAM1, respectively, while miR-126 overexpression reduced VCAM1 and CCL2 expression by hCMEC/D3 cells, suggesting that these miRs regulate leukocyte adhesion by modulating the expression of adhesion-associated endothelial mRNA targets. Hence, human brain endothelial miR-126 and miR-126* could be used as a therapeutic tool to reduce leukocyte adhesion and thus reduce neuroinflammation

Targeted disruption of tyrosylprotein sulfotransferase-2, an enzyme that catalyzes post-translational protein tyrosine O-sulfation, causes male infertility.

Tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 and -2) expressed in all mammalian cells. Tyrosine sulfation plays an important role in the function of some known TPST substrates by enhancing protein-protein interactions. To explore the role of these enzymes in vivo and gain insight into other potential TPST substrates, TPST-2-deficient mice were generated by targeted disruption of the Tpst2 gene. Tpst2(+/-) mice appear normal and, when interbred, yield litters of normal size with a Mendelian distribution of the targeted mutation. Tpst2(-/-) mice have moderately delayed growth but appear healthy and attain normal body weight by 10 weeks of age. In contrast to Tpst1(-/-) males that have normal fertility, Tpst2(-/-) males are infertile. Tpst2(-/-) sperm are normal in number, morphology, and motility in normal media and appear to capacitate and undergo acrosomal exocytosis normally. However, they are severely defective in their motility in viscous media and in their ability to fertilize zona pellucida-intact eggs. Adhesion of Tpst2(-/-) sperm to the egg plasma membrane is reduced compared with wild type sperm, but sperm-egg fusion is similar or even increased. These data strongly suggest that tyrosine sulfation of unidentified substrate(s) play a crucial role in these processes and document for the first time the critical importance of post-translational tyrosine sulfation in male fertility

Magnetic resonance imaging.

<p>Contiguous coronal images were collected from wild type (WT), <i>Tpst1-/-</i> (T1), and <i>Tpst2-/-</i> (T2) females (n = 3). The tissue region comprising the right and left sublingual and submandibular glands (A) were traced and an area per slice was calculated and a total volume was determined which was then normalized to body weight in grams as described in Methods (B). Results are expressed as mean ± S.E.M. Statistical differences between groups were tested using unpaired, two-tailed <i>t</i>-tests with equal sample variance and an α ≤ 0.05. Salivary gland volume of <i>Tpst2</i> -/- mice was significantly smaller than wild type (<i>p</i> = 0.002) and <i>Tpst1-/-</i> (<i>p</i> = 0.04), respectively (indicated by an asterisk). However, the salivary gland volumes of wild type and <i>Tpst1-/-</i> mice were not statistically different (<i>p</i> = 0.57).</p

Serum T4 levels.

<p>Serum total T4 levels were determined at 5, 10, and 15 weeks of age for 4 mice randomly selected from the 10-13 mice in each experimental group. WT = wild type. T2 = <i>Tpst2-/-</i>. Results are expressed as mean ± S.E.M.</p

Salivary gland function in wild type, <i>Tpst1-/-</i>, and <i>Tpst2</i> -/- mice.

<p>Saliva was collected from age-matched wild type (WT), <i>Tpst1-/-</i> (T1), and <i>Tpst2-/-</i> (T2) males and females as described in Methods. Pilocarpine-induced salivary flow normalized to body weight in grams (A, B) and α-amylase activity normalized to protein concentration (C, D) were measured as described in Methods. All results are represented as the mean ± S.E.M. For wild type and <i>Tpst1-/-</i> males and females, n = 5. For <i>Tpst2-/-</i> males and female, n = 3 and 4, respectively. Statistical differences between groups were tested using unpaired, two-tailed <i>t</i>-tests with equal sample variance and an α ≤ 0.05. An asterisk indicates <i>p</i> < 0.01 compared to wild type values.</p

Sulfotyrosine Western blot analysis of salivary glands.

<p>Submandibular, sublingual, and parotid glands from wild type (WT), <i>Tpst1-/-</i> (T1), and <i>Tpst2-/-</i> (T2) mice were collected, homogenized and post-nuclear supernatant were prepared as described in Methods. Non-reduced proteins (10 µg per lane) were resolved in 4-12% Bis-Tris polyacrylamide gels. Proteins were either transferred onto nitrocellulose membranes and probed with PSG2 followed by HRP conjugated secondary antibody (A) or detected with Imperial protein stain (ThermoScientific) (B). Human heparin cofactor II (HCII), a known tyrosine-sulfated protein, served as a positive control.</p

Submandibular gland histology.

<p>At 15 weeks of age submandibular glands from female (top panels) or males (bottom panels) were harvested and then fixed, wax embedded, sectioned, and stained with Masson’s trichrome as described in Methods. Images are representative of analyses of the 4 mice randomly selected from the >10 mice in each experimental group in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071822#pone-0071822-g007" target="_blank">Figure 7</a>. 100 x Objective.</p

Schematic diagram of thyroid hormone rescue protocol and timing of experimental endpoints.

<p>Sexually mature <i>Tpst2+/-</i> males and females were conditioned to control chow or chow supplemented with thyroid powder for 3 weeks. Mating pairs were then established and continued on the control or experimental diet throughout pregnancy. Weanlings were then placed on the same diet until they were sacrificed at 15 weeks of age. All animals were weighed weekly and serum total T4 levels were determined at 5, 10, and 15 weeks of age. Saliva was collected at 12 weeks of age and animals were sacrificed at 15 weeks of age for histological analysis. Groups of 10-13 animals of each genotype (Tpst2+/+, <i>Tpst2+/-</i>, <i>Tpst2-/-</i>) and sex were included in the study.</p

Body weights.

<p>Body weight were measured weekly for all mice in each experimental group (n = 10-13). WT = wild type. T2 = <i>Tpst2-/-</i>. Results are expressed as mean ± S.E.M. Differences between groups was assessed with a two-way repeated-measures ANOVA using Prism 6 software. Statistical differences at each age were tested <i>post-hoc</i> using unpaired, two-tailed <i>t</i>-tests with equal sample variance and an α ≤ 0.05.</p