Biology subjects

Egbert, J. R.

Publications and source records attributed to Egbert, J. R..

6 recordsLinked to original sources

bioRxiv2023

Phosphatases modified by LH signaling in rat ovarian follicles and their role in regulating the NPR2 guanylyl cyclase

In response to luteinizing hormone, multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-minute exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the PPP family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4-10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/544636v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@2622cdorg.highwire.dtl.DTLVardef@1b6eef7org.highwire.dtl.DTLVardef@102da9borg.highwire.dtl.DTLVardef@5e3221_HPS_FORMAT_FIGEXP M_FIG C_FIG Summary sentenceQuantitative mass spectrometric analysis of phosphatases whose phosphorylation state is rapidly modified by luteinizing hormone provides clues about how LH signaling dephosphorylates NPR2 as well as a resource for future studies.

cell biology↗

bioRxiv2021

A CRISPR toolbox for generating intersectional genetic mice for functional, molecular, and anatomical circuit mapping

BackgroundA full understanding of circuits and cellular mechanisms governing health and disease requires the dissection and multi-faceted study of discrete cell subtypes in developing and adult animal models. Recombinase-driven expression of transgenic response alleles represents a significant and powerful approach to delineate cell populations for functional, molecular, and anatomical study. In addition to single recombinase systems, the expression of two recombinases in distinct, but partially overlapping, populations allow for more defined target expression. Although the application of this method is becoming increasingly popular, the expense and difficulty associated with production of customized intersectional mouse lines have limited widespread application to more common allele manipulations that are often commercially produced at great expense. ResultsWe present a simplified CRISPR toolkit for rapid, inexpensive, and facile intersectional allele production. Briefly, we produced 7 intersectional mouse lines using a dual recombinase system, one mouse line with a single recombinase system, and three embryonic stem (ES) cell lines that are designed to study how functional, molecular, and anatomical features relate to each other in building circuits that underlie physiology and behavior. As a proof-of-principle, we applied three of these lines to different neuronal populations for anatomical mapping and functional in vivo investigation of respiratory control. We also generated a mouse line with a single recombinase-responsive allele that controls the expression of the calcium sensor Twitch-2B. This mouse line was applied globally to study the effects of follicle stimulating hormone (FSH) and luteinizing hormone (LH) on calcium release in the ovarian follicle. ConclusionsLines presented here are representative examples of outcomes possible with the successful application of our genetic toolkit for the facile development of diverse, modifiable animal models. This toolkit will allow labs to create single or dual recombinase effector lines easily for any cell population or subpopulation of interest when paired with the appropriate Cre and FLP recombinase mouse lines or viral vectors. We have made our tools and derivative intersectional mouse and ES cell lines openly available for non-commercial use through publicly curated repositories for plasmid DNA, ES cells, and transgenic mouse lines.

genetics↗

bioRxiv2020

The phosphatase inhibitor LB-100 acts synergistically with the NPR2 agonist BMN-111 to improve bone growth

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP (cGMP) in chondrocytes and severe short stature, causing achondroplasia (ACH) and acrosomelic dysplasia type Maroteaux, respectively. Previously we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+), and that in control growth plate chondrocytes, FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein. Here we tested whether a phosphatase inhibitor (LB-100) could enhance bone growth in ACH. In ex vivo imaging experiments using a FRET sensor to measure cGMP production in chondrocytes of living tibias from newborn mice, LB-100 counteracts the FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, LB-100 in combination with BMN-111 increases the rate of femur growth by [~]25% vs BMN-111 alone, restores chondrocyte terminal differentiation, increases the proliferative growth plate area of the femur, and reduces the activity of the MAP kinase pathway. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/288589v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1d0f5d3org.highwire.dtl.DTLVardef@1bb0724org.highwire.dtl.DTLVardef@1d206d1org.highwire.dtl.DTLVardef@1bd9ad7_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2019

Follicle-stimulating hormone and luteinizing hormone increase Ca2+ in the granulosa cells of mouse ovarian follicles

Summary sentenceBoth FSH and LH increase Ca2+ in the granulosa cells of intact ovarian follicles from mice expressing genetically encoded sensors.\n\nABSTRACTIn mammalian ovarian follicles, follicle stimulating hormone (FSH) and luteinizing hormone (LH) signal primarily through the G-protein Gs to elevate cAMP, but both of these hormones can also elevate Ca2+ under some conditions. Here we investigate FSH- and LH-induced Ca2+ signaling in intact follicles of mice expressing genetically encoded Ca2+ sensors, Twitch-2B and GCaMP6s. At a physiological concentration (1 nM), FSH elevates Ca2+ within the granulosa cells of preantral and antral follicles. The Ca2+ rise begins several minutes after FSH application, peaks at ~10 minutes, remains above baseline for another ~10 minutes, and depends on extracellular Ca2+. However, suppression of the FSH-induced Ca2+ increase by reducing extracellular Ca2+ does not inhibit FSH-induced phosphorylation of MAP kinase, estradiol production, or the acquisition of LH responsiveness. Like FSH, LH also increases Ca2+, when applied to preovulatory follicles. At a physiological concentration (10 nM), LH elicits Ca2+ oscillations in a subset of cells in the outer mural granulosa layer. These oscillations continue for at least 6 hours and depend on the activity of Gq family G-proteins. Suppression of the oscillations by Gq inhibition does not inhibit meiotic resumption, but does slightly attenuate ovulation. In summary, both FSH and LH increase Ca2+ in the granulosa cells of intact follicles, but the functions of these Ca2+ rises are only starting to be identified.

cell biology↗

bioRxiv2017

Dephosphorylation of the NPR2 guanylyl cyclase contributes to inhibition of bone growth by fibroblast growth factor

Activating mutations in fibroblast growth factor (FGF) receptor 3 and inactivating mutations in the NPR2 guanylyl cyclase cause similar forms of dwarfism, but how these two signaling systems interact to regulate bone growth is poorly understood. Here, by use of a mouse model in which NPR2 cannot be dephosphorylated, we show that bone elongation is opposed when NPR2 is dephosphorylated and thus produces less cyclic GMP. By developing an in vivo imaging system to measure cyclic GMP levels in intact tibia, we show that FGF-induced dephosphorylation of NPR2 decreases its guanylyl cyclase activity in growth plate chondrocytes in living bone. Thus FGF signaling lowers cyclic GMP in the growth plate, which counteracts bone elongation. These results define a new component of the signaling network by which activating mutations in the FGF receptor inhibit bone growth.

developmental biology↗

bioRxiv2017

Dephosphorylation is the Mechanism of Fibroblast Growth Factor Inhibition of Guanylyl Cyclase-B

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations of guanylyl cyclase-B (GC-B, also called NPRB or NPR2) cause dwarfism. FGF exposure inhibits GC-B activity in a chondrocyte cell line, but the mechanism of the inactivation is not known. Here, we report that FGF exposure causes dephosphorylation of GC-B in rat chondrosarcoma cells, which correlates with a rapid, potent and reversible inhibition of C-type natriuretic peptide-dependent activation of GC-B. Cells expressing a phosphomimetic mutant of GC-B that cannot be inactivated by dephosphorylation because it contains glutamate substitutions for all known phosphorylation sites showed no decrease in GC-B activity in response to FGF. We conclude that FGF rapidly inactivates GC-B by a reversible dephosphorylation mechanism, which may contribute to the signaling network by which activated FGFR3 causes dwarfism.\n\nHighlightsO_LIGuanylyl Cyclase-B is expressed in rat chondrosarcoma cells\nC_LIO_LIFGF2 induces a rapid, potent, and reversible inhibition of GC-B\nC_LIO_LIFGF2 treatment causes GC-B dephosphorylation\nC_LIO_LIFGF2 does not inhibit a dephosphorylation-resistant form of GC-B\nC_LIO_LIDephosphorylation is the mechanism of FGF2-dependent inhibition of GC-B\nC_LI\n\nAbbreviations

biochemistry↗