A G protein-coupled, IP3/protein kinase C pathway controlling the synthesis of phosphaturic hormone FGF23

Dysregulated actions of bone-derived phosphaturic hormone fibroblast growth factor 23 (FGF23) result in several inherited diseases, such as X-linked hypophosphatemia (XLH), and contribute substantially to the mortality in kidney failure. Mechanisms governing FGF23 production are poorly defined. We herein found that ablation of the Gq/11α–like, extralarge Gα subunit (XLαs), a product of GNAS, exhibits FGF23 deficiency and hyperphosphatemia in early postnatal mice (XLKO). FGF23 elevation in response to parathyroid hormone, a stimulator of FGF23 production via cAMP, was intact in XLKO mice, while skeletal levels of protein kinase C isoforms α and δ (PKCα and PKCδ) were diminished. XLαs ablation in osteocyte-like Ocy454 cells suppressed the levels of FGF23 mRNA, inositol 1,4,5-trisphosphate (IP3), and PKCα/PKCδ proteins. PKC activation in vivo via injecting phorbol myristate acetate (PMA) or by constitutively active Gqα-Q209L in osteocytes and osteoblasts promoted FGF23 production. Molecular studies showed that the PKC activation–induced FGF23 elevation was dependent on MAPK signaling. The baseline PKC activity was elevated in bones of Hyp mice, a model of XLH. XLαs ablation significantly, but modestly, reduced serum FGF23 and elevated serum phosphate in Hyp mice. These findings reveal a potentially hitherto-unknown mechanism of FGF23 synthesis involving a G protein–coupled IP3/PKC pathway, which may be targeted to fine-tune FGF23 levels

Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies

EGFR signaling mediates the chemotactic effect of PTH on mesenchymal progenitors.

<p>(A) Conditioned media from PTH-treated UMR 106-01 cells increased the phosphorylation of EGFR in mesenchymal progenitors. In this experiment, mesenchymal progenitors were treated with conditioned media for 5 min and then lysed for Western blot. (B) The enhanced phosphorylation of Akt and p38MAPK in mesenchymal progenitors by conditioned media from PTH-treated UMR106-01 cells is dependent on the EGFR pathway. Mesenchymal progenitors were pre-incubated with either DMSO or gefitinib (10 µM, GEF) for 30 min followed by addition of conditioned media to the culture. Cell lysates were collected 5 min later for Western blot analyses. (C) The EGFR inhibitor PD153035 (10 µM, PD) was added to both the upper and lower wells of the chemotaxis assay and partially blocked the PTH-induced chemotactic activity of conditioned media from UMR 106-01 cells. ***: p<0.001 vs. DMSO CON; &: p<0.01 vs. DMSO PTH. (D) An EGFR neutralizing antibody (4 µg/ml) was mixed with mesenchymal progenitors before the chemotaxis assay and suppressed the migration of mesenchymal progenitors towards conditioned media from PTH-treated UMR 106-01 cells. IgG: isotype control. **: p<0.01; ***: p<0.001 vs. CON; $: p<0.05; #: p<0.001 vs. PTH. (E) qRT-PCR demonstrates the knockdown of EGFR mRNA levels in mesenchymal progenitors by siRNAs. ***: p<0.001 vs. MOCK. (F) Immunoblotting reveals that the EGFR protein level was dramatically decreased in mesenchymal progenitors transfected with siRNAs for EGFR. (G) Blocking of EGFR expression in mesenchymal progenitors by siRNAs abolished the chemotactic migration of these cells toward conditioned media from PTH-treated UMR 106-01 cells. ***: p<0.001 vs. mock CON; #: p<0.001 vs. mock PTH.</p

A model for PTH-induced mesenchymal progenitor migration in bone.

<p>A model for PTH-induced mesenchymal progenitor migration in bone.</p

Conditioned media from PTH-treated osteoblastic cells contain chemotactic factors for bone marrow mesenchymal progenitors.

<p>(A) Migration of freshly flushed rat bone marrow cells toward PTH and conditioned media from UMR 106-01 cells. Bone marrow cells flushed from rat long bones were immediately seeded in the upper chamber of transwell plates. The bottom wells were loaded with media alone (αMEM), media containing 10 nM PTH (PTH), or conditioned media (CM) collected from UMR106-01 cells that had been treated with control (CON) or 10 nM PTH (PTH) for 4 hr. The number of cells that migrated to the bottom wells was counted 24 hr later. αMEM containing 5% FBS (FBS) was used as a positive control for cell migration. *: p<0.05; **: p<0.01 vs. αMEM. (B) Conditioned media from various PTH-treated osteoblastic and osteocytic cells stimulated the migration of either human or rat mesenchymal progenitors (MP) in the Boyden chamber assay. The cells seeded in the upper wells are depicted at the top and conditioned media loaded in the lower wells are shown at the bottom. M: αMEM. ***: p<0.001 vs. αMEM; &: p<0.01; #: p<0.001 vs. CON. (C) Microscopic images of the mesenchymal progenitors that migrated to the lower sides of filters in the assay depicted in B. (D) The migration of mesenchymal progenitors stimulated by conditioned media from PTH-treated osteoblasts is not chemokinesis. Mesenchymal progenitors were suspended in conditioned media harvested from either control or PTH-treated UMR 106-01 cells and seeded in the upper chambers. The lower wells were filled with conditioned media, resulting in a total of 4 combination types. **: p<0.01 vs. CON/CON. (E) Time course of the release of chemotactic factor(s) from osteoblasts after PTH treatment. UMR 106-01 cells were treated with PTH and conditioned media were harvested at indicated time points and loaded in the lower wells for chemotaxis assays. ***: p<0.001 vs. time 0 hr. (F) The dosage effects of PTH on chemotactic factor(s) release from osteoblasts. UMR 106-01 cells were treated with different doses of PTH for 4 hr and then conditioned media were harvested for chemotaxis assays. *: p<0.05; **: p = 0.06; ***: p<0.001 vs. 0 nM.</p

PTH stimulates the release of amphiregulin from osteoblasts to promote mesenchymal progenitor migration.

<p>(A) EGF-like ligands are chemotactic factors for mesenchymal progenitors. αMEM containing various amounts of EGF-like ligands was added in the lower wells of chemotaxis assays using rat mesenchymal progenitors. αMEM containing 5% FBS was used as positive control. *: p<0.05; **: p<0.01; ***: p<0.001 vs. CON. (B) Addition of GM6001 (10 µM, GM) in the chemotaxis assay blocked the migration of mesenchymal progenitors toward conditioned media from PTH-treated UMR 106-01 cells. ***: p<0.001 vs. CON DMSO; #: p<0.001 vs. PTH DMSO. (C) qRT-PCR shows that PTH (10 nM) induced the expression of amphiregulin in osteocytic Ocy491 cells at 1 h. (D) qRT-PCR demonstrates the knockdown of amphiregulin mRNA in UMR 106-01 cells after 1 hr of PTH (10 nM) treatment by siRNAs. **: p<0.01 vs. mock1. (E) Chemotaxis assays reveal that PTH did not stimulate the release of chemotactic factor(s) from UMR106-01 cells transfected with siRNAs for amphiregulin. ***: p<0.001 vs. CON CM; #; p<0.001 vs PTH CM mock1. (F) Amphiregulin (AR) stimulated Akt and p38MAPK phosphorylation in mesenchymal progenitors as shown by immunoblotting.</p

PI3K/Akt and p38MAPK pathways are required for the migration of mesenchymal progenitors toward conditioned media from PTH-treated osteoblastic cells.

<p>(A) Chemotaxis assays were performed with mesenchymal progenitors and conditioned media from either control- or PTH-treated UMR 106-01 cells in the presence or absence of pathway-specific inhibitors. D: DMSO; U: U0126 (20 µM); WT: wortmannin (3 µM); SB: SB202190 (20 µM). Inhibitors were added to both upper and bottom chambers. ***: p<0.001 vs. CON CM D; #: p<0.001 vs. PTH CM D. (B) Conditioned media from PTH-treated UMR 106-01 cells stimulated the phosphorylation of Akt and p38MAPK in MSCs. (C) PTH alone did not activate Akt and p38MAPK pathways in mesenchymal progenitors.</p

Mesenchymal progenitors express EGFR.

<p>(A) Flow cytometry analyses demonstrate that cultured mesenchymal progenitors (MPs) but not freshly isolated bone marrow mononuclear cells (BMMCs) express EGFR surface antigen. Blue curve: EGFR antibody; red curve: isotype control. (B) Saturation curve of binding of ¹²⁵I-EGF to mesenchymal progenitors.</p

PTH itself is not a chemotactic factor for mesenchymal progenitors.

<p>(A) qRT-PCR quantification of mRNA levels of PTH1R in UMR106-01 cells and rat mesenchymal progenitors in culture. ***: p<0.001 vs. UMR. (B) The expression of PTH1R in rat calvarial osteoblasts increases dramatically during their osteogenic differentiation as measured by qRT-PCR. PRO: proliferation stage; DIF: differentiation stage; MIN: mineralization stage. **: p<0.01; ***: p<0.001 vs. PRO. (C) PTH stimulates cAMP production in UMR 106-01 cells but not mesenchymal progenitors. ***: p<0.001 vs. con. (D) PTH alone does not stimulate the migration of mesenchymal progenitors. Chemotaxis assays were performed with lower wells filled with αMEM, αMEM containing 10 nM PTH, conditioned media from control- and PTH-treated UMR 106-01 cells, and αMEM containing 5% FBS. ***: p<0.001 vs. CON.</p