The Pathogenic TSH β-Subunit Variant C105Vfs114X Causes a Modified Signaling Profile at TSHR

1) Background: Central congenital hypothyroidism (CCH) is a rare endocrine disorder that can be caused by mutations in the β-subunit of thyrotropin (TSHB). The TSHB mutation C105Vfs114X leads to isolated thyroid-stimulating-hormone-(TSH)-deficiency and results in a severe phenotype. The aim of this study was to gain more insight into the underlying molecular mechanism and the functional effects of this mutation based on two assumptions: a) the three-dimensional (3D) structure of TSH should be modified with the C105V substitution, and/or b) whether the C-terminal modifications lead to signaling differences. 2) Methods: wild-type (WT) and different mutants of hTSH were generated in human embryonic kidney 293 cells (HEK293 cells) and TSH preparations were used to stimulate thyrotropin receptor (TSHR) stably transfected into follicular thyroid cancer cells (FTC133-TSHR cells) and transiently transfected into HEK293 cells. Functional characterization was performed by determination of Gs, mitogen activated protein kinase (MAPK) and Gq/11 activation. 3) Results: The patient mutation C105Vfs114X and further designed TSH mutants diminished cyclic adenosine monophosphate (cAMP) signaling activity. Surprisingly, MAPK signaling for all mutants was comparable to WT, while none of the mutants induced PLC activation. 4) Conclusion: We characterized the patient mutation C105Vfs114X concerning different signaling pathways. We identified a strong decrease of cAMP signaling induction and speculate that this could, in combination with diverse signaling regarding the other pathways, accounting for the patient's severe phenotype

FSHR Trans-Activation and Oligomerization

Follicle stimulating hormone (FSH) plays a key role in human reproduction through, among others, induction of spermatogenesis in men and production of estrogen in women. The function FSH is performed upon binding to its cognate receptor—follicle-stimulating hormone receptor (FSHR) expressed on the surface of target cells (granulosa and Sertoli cells). FSHR belongs to the family of G protein-coupled receptors (GPCRs), a family of receptors distinguished by the presence of various signaling pathway activation as well as formation of cross-talking aggregates. Until recently, it was claimed that the FSHR occurred naturally as a monomer, however, the crystal structure as well as experimental evidence have shown that FSHR both self-associates and forms heterodimers with the luteinizing hormone/chorionic gonadotropin receptor—LHCGR. The tremendous gain of knowledge is also visible on the subject of receptor activation. It was once thought that activation occurs only as a result of ligand binding to a particular receptor, however there is mounting evidence of trans-activation as well as biased signaling between GPCRs. Herein, we describe the mechanisms of aforementioned phenomena as well as briefly describe important experiments that contributed to their better understanding

REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering

Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient.</p

Enhanced LH action in transgenic female mice expressing hCGβ-subunit induces pituitary prolactinomas; the role of high progesterone levels

The etiology of pituitary adenomas remains largely unknown, with the exception of involvement of estrogens in the formation of prolactinomas. We have examined the molecular pathogenesis of prolactin-producing pituitary adenomas in transgenic female mice expressing the human choriongonadotropin (hCG) β-subunit. The LH/CG bioactivity is elevated in the mice, with consequent highly stimulated ovarian progesterone (P4) production, in the face of normal estrogen secretion. Curiously, despite normal estrogen levels, large prolactinomas developed in these mice, and we provide here several lines of evidence that the elevated P4 levels are involved in the growth of these estrogen-dependent tumors. The antiprogestin mifepristone inhibited tumor growth, and combined postgonadectomy estradiol/P4 treatment was more effective than estrogen alone in inducing tumor growth. Evidence for direct growth-promoting effect of P4 was obtained from cultures of primary mouse pituitary cells and rat somatomammotroph GH3 cells. The mouse tumors and cultured cells revealed stimulation of the cyclin D1/cyclin-dependent kinase 4/retinoblastoma protein/transcription factor E2F1 pathway in the growth response to P4. If extrapolated to humans, and given the importance of endogenous P4 and synthetic progestins in female reproductive functions and their pharmacotherapy, it is relevant to revisit the potential role of these hormones in the origin and growth of prolactinomas

The Luteinizing Hormone Receptor Knockout Mouse as a Tool to Probe the in Vivo Actions of Gonadotropic Hormones/Receptors in Females

Mouse models with altered gonadotropin functions have provided invaluable insight into the functions of these hormones/receptors. Here we describe the repurposing of the infertile and hypogonadal luteinizing hormone receptor (LHR) knockout mouse model (LuRKO), to address outstanding questions in reproductive physiology. Using crossbreeding strategies and physiological and histological analyses, we first addressed the physiological relevance of forced LHR homomerization in female mice using BAC expression of 2 ligand-binding and signaling deficient mutant LHR, respectively, that have previously shown to undergo functional complementation and rescue the hypogonadal phenotype of male LuRKO mice. In female LuRKO mice, coexpression of signaling and binding deficient LHR mutants failed to rescue the hypogonadal and anovulatory phenotype. This was apparently due to the low-level expression of the 2 mutant LHR and potential lack of luteinizing hormone (LH)/LHR-dependent pleiotropic signaling that has previously been shown at high receptor densities to be essential for ovulation. Next, we utilized a mouse model overexpressing human chorionic gonadotropin (hCG) with increased circulating "LH/hCG"-like bioactivity to ~40 fold higher than WT females, to determine if high circulating hCG in the LuRKO background could reveal putative LHR-independent actions. No effects were found, thus, suggesting that LH/hCG mediate their gonadal and non-gonadal effects solely via LHR. Finally, targeted expression of a constitutively active follicle stimulating hormone receptor (FSHR) progressed antral follicles to preovulatory follicles and displayed phenotypic markers of enhanced estrogenic activity but failed to induce ovulation in LuRKO mice. This study highlights the critical importance and precise control of functional LHR and FSHR for mediating ovarian functions and of the potential repurposing of existing genetically modified mouse models in answering outstanding questions in reproductive physiology. © 2021 The Author(s). Published by Oxford University Press on behalf of the Endocrine Society.</p

IGIST - A Kinetic Bioassay for Pertussis Toxin Based on Its Effect on Inhibitory GPCR Signaling

Detection of pertussis toxin (PTX) activity is instrumental for the development and manufacturing of pertussis vaccines. These quality and safety measures require thousands of mice annually. Here, we describe Interference in Gαi-mediated Signal Transduction (iGIST), an animal-free kinetic bioassay for detection of PTX, by measuring its effect on inhibitory G protein-coupled receptor (GPCR) signaling. PTX ADP-ribosylates inhibitory α-subunits of the heterotrimeric G proteins, thereby perturbing the inhibitory GPCR signaling. iGIST is based on HEK293 cells coexpressing a somatostatin receptor 2 (SSTR2), which is an inhibitory GPCR controllable by a high-affinity agonist octreotide; and a luminescent 3′5′-cyclic adenosine monophosphate (cAMP) probe. iGIST has a low sensitivity threshold in the pg/mL range of PTX, surpassing by 100-fold in a parallel analysis the currently used in vitro end-point technique to detect PTX, the cluster formation assay (CFA) in Chinese hamster ovary cells. iGIST also detects PTX in complex samples, i.e., a commercial PTX-toxoid-containing pertussis vaccine that was spiked with an active PTX. iGIST has an objective digital readout and is observer independent, offering prospects for automation. iGIST emerges as a promising animal-free alternative to detect PTX activity in the development and manufacturing of pertussis vaccines. iGIST is also expected to facilitate basic PTX research, including identification and characterization of novel compounds interfering with PTX.</p

In vitro Targetability Validation of Peptide-Functionalized Mesoporous Silica Nanoparticles in the Presence of Serum Proteins

Demonstration of receptor-mediated targeting of nanoparticles to specific organs and/or cell types is an integral aim in many bionanomedicine development projects. However, engagement of targeted receptors with ligands on nanocarriers, which is the cornerstone of the active targeting concept, is challenging to study under biologically relevant conditions and thus often stays overlooked. In this work, we utilize an in-house established bioassay for in vitro targetability validation of mesoporous silica nanoparticles (MSNs), functionalized with high-affinity peptide ligands to somatostatin receptors via protective group chemistry, ensuring the correct orientation of the peptide's pharmacophore. We demonstrate that targeted nanoparticles, but not scrambled peptide-decorated counterparts, specifically engage the targeted receptors in living cells in culture media containing serum protein. The importance of being able to exclude false positives originating from the premature detachment of targeting peptides from the MSNs is highlighted

PIM kinases inhibit AMPK activation and promote tumorigenicity by phosphorylating LKB1

BackgroundThe oncogenic PIM kinases and the tumor-suppressive LKB1 kinase have both been implicated in the regulation of cell growth and metabolism, albeit in opposite directions. Here we investigated whether these kinases interact with each other to influence AMPK activation and tumorigenic growth of prostate and breast cancer cells.MethodsWe first determined how PIM and LKB1 kinases affect AMPK phosphorylation levels. We then used in vitro kinase assays to demonstrate that LKB1 is phosphorylated by PIM kinases, and site-directed mutagenesis to identify the PIM target sites in LKB1. The cellular functions of PIM and LKB1 kinases were evaluated using either pan-PIM inhibitors or CRISPR/Cas9 genomic editing, with which all three PIM family members and/or LKB1 were knocked out from PC3 prostate and MCF7 breast cancer cell lines. In addition to cell proliferation assays, we examined the effects of PIM and/or LKB1 loss on tumor growth using the chick embryo chorioallantoic membrane (CAM) xenograft model.ResultsWe provide both genetic and pharmacological evidence to demonstrate that inhibition of PIM expression or activity increases phosphorylation of AMPK at Thr172 in both PC3 and MCF7 cells, but not in their derivatives lacking LKB1. This is explained by our observation that all three PIM family kinases can phosphorylate LKB1 at Ser334. Wild-type LKB1, but not its phosphodeficient derivative, can restore PIM inhibitor-induced AMPK phosphorylation in LKB1 knock-out cells. In the CAM model, loss of LKB1 enhances tumorigenicity of PC3 xenografts, while cells lacking both LKB1 and PIMs exhibit slower proliferation rates and form smaller tumors.ConclusionPIM kinases are novel negative regulators of LKB1 that affect AMPK activity in an LKB1-dependent fashion. The impairment of cell proliferation and tumor growth in cells lacking both LKB1 and PIMs indicates that these kinases possess a shared signaling role in the context of cancer. These data also suggest that PIM inhibitors may be a rational therapeutic option for LKB1-deficient tumors.</p