The prolactin receptor (PRLR) signals predominantly through the JAK2-STAT5 pathway regulating multiple physiological functions relating to fertility, lactation, and metabolism. However, the molecular pathology and role of PRLR mutations and signalling are incompletely defined, with progress hampered by a lack of reported disease-associated PRLR variants. To date, two common germline PRLR variants are reported to demonstrate constitutive activity, with one, Ile146Leu, overrepresented in benign breast disease, while a rare activating variant, Asn492Ile, is reported to be associated with an increased incidence of prolactinoma. In contrast, an inactivating germline heterozygous PRLR variant (His188Arg) was reported in a kindred with hyperprolactinaemia, while an inactivating compound heterozygous PRLR variant (Pro269Leu/Arg171Stop) was identified in an individual with hyperprolactinaemia and agalactia. We hypothesised that additional rare germline PRLR variants, identified from large-scale sequencing projects (ExAC and GnomAD), may be associated with altered in vitro PRLR signalling activity. We therefore evaluated >300 previously uncharacterised non-synonymous, germline PRLR variants and selected 10 variants for in vitro analysis based on protein prediction algorithms, proximity to known functional domains and structural modelling. Five variants, including extracellular and intracellular domain variants, were associated with altered responses when compared to the wild-type receptor. These altered responses included loss- and gain-of-function activities related to STAT5 signalling, Akt and FOXO1 activity, as well as cell viability and apoptosis. These studies provide further insight into PRLR structure–function and indicate that rare germline PRLR variants may have diverse modulating effects on PRLR signalling, although the pathophysiologic relevance of such alterations remains to be defined.
Caroline M Gorvin, Paul J Newey, and Rajesh V Thakker
Rishel B Vohnoutka, Annapurna Kuppa, Yash Hegde, Yue Chen, Asmita Pant, Maurice E Tohme, Eun-Young (Karen) Choi, Sean M McCarty, Devika P Bagchi, Xiaomeng Du, Yanhua Chen, Vincent L Chen, Hiroyuki Mori, Lawrence F Bielak, Lillias H Maguire, Samuel K Handelman, Jonathan Z Sexton, Thomas L Saunders, Brian D Halligan, and Elizabeth K Speliotes
Human genome-wide association studies found single-nucleotide polymorphisms (SNPs) near LYPLAL1 (Lysophospholipase-like protein 1) that have sex-specific effects on fat distribution and metabolic traits. To determine whether altering LYPLAL1 affects obesity and metabolic disease, we created and characterized a mouse knockout (KO) of Lyplal1. We fed the experimental group of mice a high-fat, high-sucrose (HFHS) diet for 23 weeks, and the controls were fed regular chow diet. Here, we show that CRISPR-Cas9 whole-body Lyplal1 KO mice fed an HFHS diet showed sex-specific differences in weight gain and fat accumulation as compared to chow diet. Female, not male, KO mice weighed less than WT mice, had reduced body fat percentage, had white fat mass, and had adipocyte diameter not accounted for by changes in the metabolic rate. Female, but not male, KO mice had increased serum triglycerides, decreased aspartate, and decreased alanine aminotransferase. Lyplal1 KO mice of both sexes have reduced liver triglycerides and steatosis. These diet-specific effects resemble the effects of SNPs near LYPLAL1 in humans, suggesting that LYPLAL1 has an evolutionary conserved sex-specific effect on adiposity. This murine model can be used to study this novel gene-by-sex-by-diet interaction to elucidate the metabolic effects of LYPLAL1 on human obesity.
Sarah Theresa Boyle
The tumor microenvironment is a dynamic ecosystem of stromal and immune cells that, under the influence of cancer cells, govern biochemical signaling, mechanical signaling via production and remodeling of the extracellular matrix (ECM), formation of vascular networks, and ultimately promotion of tumor growth. In breast cancer, hormone receptor-mediated signaling is a key coordinator of cancer cell proliferation and invasiveness not only through cell-autonomous means but also via cancer cell–stroma cross-talk. In the absence of hormone receptors, a different microenvironment landscape emerges, which comes with its own challenges for therapy. This review summarizes the current knowledge regarding the associations of hormone receptor profiles with composition of the microenvironment, how hormones directly influence stromal cells, immune cells and cells associated with the vasculature, and the paracrine mechanisms that lead to the formation of a tumor-promoting ECM.
Rikus Botha, Shree Senthil Kumar, Natasha L Grimsey, and Kathleen Grace Mountjoy
The human melanocortin-4 receptor plays a critical role regulating energy homeostasis. Studies on obesogenic hMC4R variants have not yet revealed how human melanocortin-4 receptor maintains body weight. Here, we identified a signaling profile for obesogenic constitutively active H76R and L250Q human melanocortin-4 receptor variants transfected in HEK293 cells that included constitutive activity for adenylyl cyclase, cyclic adenosine monophosphate response element-driven transcription, and calcium mobilization, but not phosphorylated extracellular signal-regulated kinase 1/2 activity. Importantly, the signaling profile included impaired α-melanocyte-stimulating-hormone-induced cyclic adenosine monophosphate response element-driven transcription, but not impaired α-melanocyte-stimulating-hormone-induced adenylyl cyclase, calcium, or phosphorylated extracellular signal-regulated kinase 1/2. This profile was not observed for transfected H158R, a constitutively active human melanocortin-4 receptor variant associated with overweight, but not obesity. We concluded that there is potential for α-melanocyte-stimulating-hormone-induced cyclic adenosine monophosphate response element-driven transcription in HEK293 cells transfected with obesogenic human melanocortin-4 receptor variants to be the key predictive tool for determining whether they exhibit loss-of-function. Furthermore, in vivo α-melanocyte-stimulating-hormone-induced human melanocortin-4 receptor cyclic adenosine monophosphate response element-driven transcription may be key for maintaining body weight.
Parmita Kar and Ravinder Goswami
Basal ganglia calcification (BGC) is a common complication in hypoparathyroid patients, linked to hyperphosphatemia and altered vitamin-D and calcium homeostasis following conventional therapy. The pathogenesis of BGC in hypoparathyroidism is not clear. Recently, we developed an ex vivo model of BGC using rat-striatal cell culture in 10.0 mmol/L of β-glycerophosphate (31.8 mg/dL phosphate). However, the effect of 1,25(OH)2 D, calcium, and milder phosphate excess on BGC in hypoparathyroidism is not known. This study describes two modified ex vivo models investigating pathogenesis of BGC in ‘drug-naïve’ and ‘conventionally treated’ hypoparathyroid state. The first modification involved striatal cells cultured in low concentration 1,25(OH)2D (16.0 pg/mL), ionized calcium(0.99 mmol/L), hPTH(1-34) (6.0 pg/mL), and 2.68 mmol/L (8.3 mg/dL) of phosphate akin to ‘drug-naïve’ state for 24 days. In second modification, striatal cells were exposed to 46.0 pg/mL of 1,25(OH)2D, normal ionized calcium of 1.17 mmol/L, and 2.20 mmol/L (6.8 mg/dL) of phosphate akin to ‘conventionally treated’ state. Striatal cell culture under ‘drug-naïve’ state showed that even 16.0 pg/mL of 1,25(OH)2D enhanced the calcification. In ‘conventionally treated’ model, striatal cell calcification was enhanced in 54% cases over ‘drug-naïve’ state. Calcification in ‘conventionally treated’ state further increased on increasing phosphate to 8.3 mg/dL, suggesting importance of phosphatemic control in hypoparathyroid patients. Striatal cells in ‘drug-naïve’ state showed increased mRNA expression of pro-osteogenic Wnt3a, Cd133,Vglut-1-neuronal phosphate-transporters, calcium-ion channel-Trvp2,Alp, and Collagen-1α and decreased expression of Ca-II. These models suggest that in ‘drug-naïve’ state, 1,25(OH)2D along with moderately elevated phosphate increases the expression of pro-osteogenic molecules to induce BGC. Although normalization of calcium in ‘conventionally treated’ state increased the expression of Opg, Osterix, Alp, and Cav2, calcification increased only in a subset, akin to variation in progression of BGC in hypoparathyroid patients on conventional therapy.
Jin Bai, Thomas J Lechuga, Joshua Makhoul, Hao Yan, Carol Major, Afshan Hameed, and Dong-bao Chen
Elevated endogenous estrogens stimulate human uterine artery endothelial cell (hUAEC) hydrogen sulfide (H2S) production by selectively upregulating the expression of H2S synthesizing enzyme cystathionine β-synthase (CBS), but the underlying mechanisms are underdetermined. We hypothesized that CBS transcription mediates estrogen-stimulated pregnancy-dependent hUAEC H2S production. Estradiol-17β (E2β) stimulated CBS but not cystathionine γ-lyase (CSE) expression in pregnant human uterine artery ex vivo, which was attenuated by the estrogen receptor (ER) antagonist ICI 182,780. E2β stimulated CBS mRNA/protein and H2S production in primary hUAEC from nonpregnant and pregnant women, but with greater responses in pregnant state; all were blocked by ICI 182,780. Human CBS promoter contains multiple estrogen-responsive elements (EREs), including one ERE preferentially binding ERα (αERE) and three EREs preferentially binding ERβ (βERE), and one full ERE (α/βERE) and one half ERE (½α/βERE) binding both ERα and ERβ. Luciferase assays using reporter genes driven by human CBS promoter with a series of 5′-deletions identified the α/βEREs binding both ERα and ERβ (α/βERE and ½α/βERE) to be important for baseline and E2β-stimulated CBS promoter activation. E2β stimulated ERα/ERβ heterodimerization by recruiting ERα to α/βEREs and βERE, and ERβ to βERE, α/βEREs, and αERE. ERα or ERβ agonist alone trans-activated CBS promoter, stimulated CBS mRNA/protein and H2S production to levels comparable to that of E2β-stimulated, while ERα or ERβ antagonist alone abrogated E2β-stimulated responses. E2β did not change human CSE promoter activity and CSE mRNA/protein in hUAEC. Altogether, estrogen-stimulated pregnancy-dependent hUAEC H2S production occurs by selectively upregulating CBS expression via ERα/ERβ-directed gene transcription.
Selina Mäkinen, Neeta Datta, Savithri Rangarajan, Yen H Nguyen, Vesa M Olkkonen, Aino Latva-Rasku, Pirjo Nuutila, Markku Laakso, and Heikki A Koistinen
Finnish-specific gene variant p.P50T/AKT2 (minor allele frequency (MAF) = 1.1%) is associated with insulin resistance and increased predisposition to type 2 diabetes. Here, we have investigated in vitro the impact of the gene variant on glucose metabolism and intracellular signalling in human primary skeletal muscle cells, which were established from 14 male p.P50T/AKT2 variant carriers and 14 controls. Insulin-stimulated glucose uptake and glucose incorporation into glycogen were detected with 2-[1,2-3H]-deoxy-D-glucose and D-[14C]-glucose, respectively, and the rate of glycolysis was measured with a Seahorse XFe96 analyzer. Insulin signalling was investigated with Western blotting. The binding of variant and control AKT2-PH domains to phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) was assayed using PIP StripsTM Membranes. Protein tyrosine kinase and serine-threonine kinase assays were performed using the PamGene® kinome profiling system. Insulin-stimulated glucose uptake and glycogen synthesis in myotubes in vitro were not significantly affected by the genotype. However, the insulin-stimulated glycolytic rate was impaired in variant myotubes. Western blot analysis showed that insulin-stimulated phosphorylation of AKT-Thr308, AS160-Thr642 and GSK3β-Ser9 was reduced in variant myotubes compared to controls. The binding of variant AKT2-PH domain to PI(3,4,5)P3 was reduced as compared to the control protein. PamGene® kinome profiling revealed multiple differentially phosphorylated kinase substrates, e.g. calmodulin, between the genotypes. Further in silico upstream kinase analysis predicted a large-scale impairment in activities of kinases participating, for example, in intracellular signal transduction, protein translation and cell cycle events. In conclusion, myotubes from p.P50T/AKT2 variant carriers show multiple signalling alterations which may contribute to predisposition to insulin resistance and T2D in the carriers of this signalling variant.
Xiaopan Yang, Xiaojing Fan, Jiangyue Feng, Tinghui Fan, Jingfei Li, Linfei Huang, Luming Wan, Huan Yang, Huilong Li, Jing Gong, Yanhong Zhang, Qi Gao, Fei Zheng, Lei Xu, Haotian Lin, Dandan Zhang, Hongbin Song, Yufei Wang, Xueping Ma, Zhiwei Sun, Cheng Cao, Xiaoli Yang, Hui Zhong, Yi Fang, and Congwen Wei
Golgi protein 73 (GP73), also called Golgi membrane protein 1 (GOLM1), is a resident Golgi type II transmembrane protein and is considered as a serum marker for the detection of a variety of cancers. A recent work revealed the role of the secreted GP73 in stimulating liver glucose production and systemic glucose homeostasis. Since exaggerated hepatic glucose production plays a key role in the pathogenesis of type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), GP73 may thus represent a potential therapeutic target for treating diabetic patients with pathologically elevated levels. Here, in this study, we found that the circulating GP73 levels were significantly elevated in T2DM and positively correlated with hemoglobin A1c. Notably, the aberrantly upregulated GP73 levels were indispensable for the enhanced protein kinase A signaling pathway associated with diabetes. In diet-induced obese mouse model, GP73 siRNA primarily targeting liver tissue was potently effective in alleviating abnormal glucose metabolism. Ablation of GP73 from whole animals also exerted a profound glucose-lowering effect. Importantly, neutralizing circulating GP73 improved glucose metabolism in streptozotocin (STZ) and high-fat diet/STZ-induced diabetic mice. We thus concluded that GP73 was a feasible therapeutic target for the treatment of diabetes.
Belinda J Petri and Carolyn M Klinge
Epitranscriptomic modification of RNA regulates human development, health, and disease. The true diversity of the transcriptome in breast cancer including chemical modification of transcribed RNA (epitranscriptomics) is not well understood due to limitations of technology and bioinformatic analysis. N-6-methyladenosine (m6A) is the most abundant epitranscriptomic modification of mRNA and regulates splicing, stability, translation, and intracellular localization of transcripts depending on m6A association with reader RNA-binding proteins. m6A methylation is catalyzed by the METTL3 complex and removed by specific m6A demethylase ALKBH5, with the role of FTO as an ‘eraser’ uncertain. In this review, we provide an overview of epitranscriptomics related to mRNA and focus on m6A in mRNA and its detection. We summarize current knowledge on altered levels of writers, readers, and erasers of m6A and their roles in breast cancer and their association with prognosis. We summarize studies identifying m6A peaks and sites in genes in breast cancer cells.