Prolactin Receptor Research Articles

In silico evaluation of isoflavones for its galactagogue activity in promoting lactation

Breastfeeding is crucial for the health of both mothers and infants, providing both short-term and long-term benefits. Breast milk contains all the necessary nutrients for both the mother and child. Unfortunately, many women are experiencing a low milk supply, resulting in infants not receiving sufficient immune support. To increase milk production, there are various herbal and pharmaceutical galactagogues available. Plants in the Caryophyllaceae family contain phytoestrogen, a compound that helps stimulate milk production. Isoflavone is one type of phytoestrogens. Isoflavones have many properties like bactericidal, fungicidal and anti-ringworm and are used to treat breast and prostate cancer and bone-related diseases. Spergula arvensis L., a member of the Caryophyllaceae family, is rich in isoflavones. Nine specific compounds were chosen due to their connection to mammary gland function. A preliminary in silico study was conducted to examine the interaction between isoflavones and prolactin receptors through molecular docking. The compound quercetin, an isoflavone, showed a better binding affinity to the receptor.

OR0304 Novel Dual Antagonists Of The Growth Hormone Receptor And The Prolactin Receptor

Abstract Disclosure: R. Basu: None. J.J. Kopchick: None. An excess of the human growth hormone (hGH) is associated with multiple pathophysiology including acromegaly, diabetes, cancer, renal dysfunction and age-associated cognitive impairment. On the other hand, a congenital lack of GH action in humans and mice render a protective effect from the above co-morbidities. Consequently, there exists significant pharmaceutical interest in developing antagonists of GH action - rendered via binding and activating the GHR, as well as the prolactin receptor (PRLR), which is particularly relevant for PRLR-rich cancers of the breast and prostate. Our laboratory has pioneered the discovery of the first and only FDA approved GHR antagonist - Somavert (pegvisomant for injection, Pfizer Inc., USA) - currently prescribed for the treatment of acromegaly. Here we present two new and unique designs of antagonists which block the complete spectrum of GH action at both GHR and PRLR binding. The first GHR antagonist design is a full-length G120K-hGH peptide at the core, incorporating targeted cysteine (single or double) substitutions at carefully selected solvent-accessible residues distal to the binding sites. To these substituted cysteines, maleimide activated discrete PEGs (MAL-dPEG; neutral or anionic, branched, different sizes) were specifically conjugated to build a library of multiple putative GHR antagonists. Binding studies and biochemical activities reveal multiple promising GHRA candidates with high binding affinities to both GHR and PRLR, while the biological assays show robust inhibition of GH driven intracellular signaling and oncogenic processes in cultured human cancer cell lines. Intraperitoneal administration of a selected candidate (compound-G) in mice significantly reduced serum IGF1 and IGFBP3 as well, confirming in vivo efficacy. The second candidate antagonist termed ‘S1H’ is a 16-amino-acid peptide designed as a direct sequence mimetic of a unique fragment of the site-1 binding helix of the hGH molecule. To this end we have characterized S1H with mass spectrometry, circular dichroism spectropolarimetry, and in vitro biological assays using human cell lines expressing GHR. S1H inhibited GH as well as PRL induced STAT5 phosphorylation in a dose-dependent manner in multiple cell lines. Moreover, using alanine scanning mutagenesis studies, we identified a strong correlation between the helical propensity and biological activity of S1H - allowing extensive structure activity relationship studies. Thus, we present two distinct designs, preparation and characterization of dual antagonists of the GH action at the level of GHR and PRLR, with prospective applications in pathologies associated with a GH excess and also as valuable compounds to study the nature of human GH-GHR interactions. We currently continue to develop both compounds for pre-clinical validations. Presentation: Thursday, June 15, 2023

FRI295 Prolactin Exerts Anti- And Pro-inflammatory Actions In Joint Tissues That Are Cytokine-Dependent

Abstract Disclosure: J.F. García Rodrigo: None. G. Ortiz: None. O. Martínez Díaz: None. X. Ruiz Herrera: None. M. Ledesma Colunga: None. G. Martínez de la Escalera: None. C. Clapp: None. The close association between rheumatoid arthritis (RA), sex, reproductive state, and stress have long-linked the sexually dimorphic, reproductive, stress-related hormone prolactin (PRL) to disease progression. However, this role is questioned by the fact that PRL has both pro-inflammatory and anti-inflammatory outcomes in RA with mechanisms that are unclear. Here, we show that PRL modifies in an opposite manner the inflammatory action of IL-1β and TNF-α in mouse synovial fibroblasts (SF) in culture and in joints in vivo. SF isolated from the knee joint of male C57BL/6 mice were incubated with or without IL-1β (1 ng/mL) or TNF-α (10 ng/mL) in the presence or absence of PRL (2.3 μg/mL). Both, IL-1β and TNF-α upregulated the metabolic activity (MTT assay) and the RT-qPCR expression of pro-inflammatory mediators (IL-1β, IL-6, iNOS) via the activation of the NF-κB signaling pathway (immunocytofluorescence, Western blot). However, IL-1β increased and TNF-α decreased the levels of the long PRL receptor isoform (RT-qPCR, Western blot) and this opposite action associated with contrasting effects of PRL in these cells. PRL decreased the IL-1β upregulation of metabolic activity and expression of pro-inflammatory mediators by blocking the degradation of NF-κB inhibitors kB-beta (Iκ-Bβ) and alpha (Iκ-Bα) and, thereby, interfering with the nuclear translocation of NF-κB. By contrast, PRL increased TNF-α-induced stimulation of metabolic activity and expression of pro-inflammatory mediators by promoting the nuclear translocation of NF-κB led by the increased degradation of Iκ-Bβ and Iκ-Bα. The double-faceted regulatory role of PRL against the two cytokines also manifested in vivo. IL-1β (80 ng) or TNF-α (300 ng) with or without PRL (2 μg) were injected into the intra-articular space of the knee joint of mice and, after 24 h, joint inflammation was monitored through the expression of pro-inflammatory mediators (IL-1β and iNOS). Both IL-1β and TNF-α upregulated the expression of IL-1β and iNOS in the joint. However, PRL inhibited the joint proinflammatory effect of IL-1β on IL-1β and iNOS, but not that of TNF-α. We conclude that the outcome of PRL actions on joint inflammation is dependent on its interaction with specific inflammatory cytokines, the level of the PRL receptor, and the activation of NF-κB signaling. We speculate that the opposite effects of PRL help balance joint inflammation in RA and that determining the mechanisms underlying dual actions provide insights into the pathophysiology of RA and the development of new treatments. Supported by UNAM (DGAPA-PAPIIT IN202321). Presentation: Friday, June 16, 2023

FRI303 PRL-R Variants Are Not Only Associated With Prolactinomas But Also With Dopamine Agonist Resistance

Abstract Disclosure: A.R. Moreira: None. E.B. Trarbach: None. A. Glezer: None. G.A. Maciel: None. C.B. Bueno: None. A.S. Monteiro: None. M. Padula: None. I.P. Grande: None. M.D. Bronstein: None. Introduction: Knockout prolactin receptor gene (PRL-R) mice are animal models for prolactinomas and PRL acts via autocrine/paracrine inhibiting lactotroph proliferation. Recent studies identified variants in PRL-R gene in patients with prolactinoma and two of them Glu400(376)Gln/p.Asn516(492)Ile, were more common in patients compared to controls. Functional analysis of the p.Asn516(492)Ile variant showed an increase in Akt expression and cell proliferation induced by PRL. However, the association between PRL-R variants and response to cabergoline (CAB) treatment has not been investigated. Objective: This study aims to analyze the PRL-R gene in an extensive cohort of patients with sporadic prolactinomas and evaluate its association with clinical, laboratorial and tumor imaging characteristics, as well as CAB treatment resistance. Methods: DNA was extracted from peripheral blood samples of 178 patients with prolactinoma and 146 were classified according to cabergoline response. The PRL-R gene was analyzed by PCR techniques and automated Sanger-type sequencing. Four in silico tools were used for analysis. Results: We found six PRL-R variants: p.Ile100(76)Val (n=18), p.Ile170(146)Leu (n=6), p.Glu400(376)Gln/p.Asn516(492)Ile (n=3), p.Glu470Asp (n =1) and p.Ala591Pro (n=1), the last two described for the first time in prolactinomas´ patients. The variants p.Glu400(376)Gln/p.Asn516(492)Ile and p.Ala591Pro were more frequent amongst patients compared to genomic databases and the p.Asn516(492)Ile showed pathogenic potential using in silico analysis. PRL-R variants were associated with male sex (P=0.015), higher serum PRL levels (P=0.007), larger tumors (P=0.001), higher frequency of macroadenomas (P=0.031) and cabergoline resistance (P<0.001). Considering together only the PRL-R variants classified as pathogenic/probably pathogenic (p.Ile170(146)Leu, p.Glu400(376)Gln/Asn516(492) and p.Ala591Pro) versus the benign variants (p.Ile100(76)Val, p.Glu470Asp and wild type) we also observed a higher levels of serum PRL (P=0.008), largest tumor diameter (P=0.028) and higher frequency of CAB resistance (P=0.048).Conclusion: Our study showed a higher frequency of the PRL-R variants p.Glu400(376)Gln/p.Asn516(492)Ile, and p.Ala591Pro in patients with prolactinomas when compared to Brazilian and international databases, suggesting that these variants could play a role in the prolactinoma tumorigenesis. Patients with PRL-R variants presented male predominance, higher serum PRL levels, larger tumor size at diagnosis and higher frequency of CAB resistance. More studies evaluating germinative and somatic PRL-R variants are needed to clarify the PRL/PRL-R system role in the tumorigenesis and progression of prolactinomas and their interesting association with CAB resistance. Presentation: Friday, June 16, 2023

Seasonal patterns of prolactin, prolactin receptor, and STAT5 expression in the ovaries of wild ground squirrels (Citellus dauricus Brandt).

Prolactin (PRL) is a hormone crucial for normal reproduction, functioning as an autocrine, paracrine, and endocrine factor. This study aimed to examine the immunolocalization and expression patterns of PRL, prolactin receptor (PRLR), and signal transducer and activator of transcription 5 (STAT5) in the ovaries of wild ground squirrels during both breeding and non-breeding periods. Significant seasonal variations were observed in ovarian weights, with higher values during the breeding season and relatively lower values during the nonbreeding season. PRL, PRLR, STAT5, and p-STAT5 were immunolocalized in granulosa cells and luteal cells during the breeding season, whereas they were exclusively found in granulosa cells during the non-breeding season. The mRNA expression levels of Prl, Prlr, and Stat5 were increased in ovarian tissues during the breeding season compared to the non-breeding season. Moreover, the mean mRNA levels of Prl, Prlr, and Stat5 exhibited a positive correlation with ovarian weights. Both circulating PRL and ovarian PRL concentrations were significantly elevated during the breeding season. Additionally, transcriptomic analysis of ovarian tissues revealed differentially expressed genes possibly associated with ovarian function and mammary gland development, including ovarian follicle development, steroid synthesis, and regulation of reproductive process. These findings suggest that PRL might play an essential endocrine, autocrine, or paracrine role in the regulation of seasonal changes in the ovarian functions in wild ground squirrels.

Associations of polymorphisms in prolactin and dopamine receptor D2 genes with reproductive traits on Silkie chicken

The Silkie chicken (Gallus gallus domesticus Brisson) is one of domestic chicken breeds with commercial rearing and breeding potentials for egg production. Prolactin (PRL) and dopamine D2 receptor (DRD2) are potential genes associated with reproductive traits in chickens. This study was conducted to analyze the association of PRL and DRD2 insertion/deletion (Indel) polymorphisms with chicken reproductive traits in Silkie chickens. A total of 380 hens from 16-40 weeks of age were used, with each one being placed in a separate cage. DNA isolation was performed using feather samples, and genotypes were detected using the Indel technique. Two polymorphisms consisting of 24 base pair (bp) Indel in the promoter region of the PRL gene and 22 bp Indel in the promoter region of the DRD2 gene were identified. At both sites, the Indel polymorphisms did not follow the Hardy-Weinberg equilibrium. In addition, with the exception of total eggs over 23 weeks of laying in the PRL gene, the analysis revealed no association between these polymorphic loci and any traits collected. In conclusion, birds with the DD genotype produced the maximum egg yield (73.6 eggs/hen), whereas those with the II genotype produced approximately 9 fewer eggs (64.1 eggs/hen), resulting in laying rates of 45.7% and 40.1%, respectively. For enhancing the egg-laying capacity of Silkie chickens via selective breeding, opting for DD birds with DD genotype of PRL Indel is highly recommended.

Limits to sustained energy intake. XXXIII. Thyroid hormones play important roles in milk production but do not define the heat dissipation limit in Swiss mice.

The limits to sustained energy intake set physiological upper boundaries that affect many aspects of human and animal performance. The mechanisms underlying these limits however remain unclear. We exposed Swiss mice to either supplementary thyroid hormones (THs) or methimazole during lactation at 21 °C or 32.5 °C, and measured food intake, resting metabolic rate (RMR), milk energy output (MEO), serum THs and mammary gland gene expression of females, and litter size and mass of their offspring. Lactating females developed hyperthyroidism following exposure to supplementary THs at 21 °C, but they did not significantly change body temperature, asymptotic food intake, RMR or MEO, and litter and mass were unaffected. Hypothyroidism, induced by either methimazole or 32.5 °C exposure, significantly decreased asymptotic food intake, RMR and MEO, resulting in significantly decreased litter size and litter mass. Furthermore, gene expression of key genes in the mammary gland were significantly decreased by either methimazole or hot exposure, including gene expression of THs and prolactin receptors, and Stat5a and Stat5b. This suggests that endogenous THs are necessary to maintain sustained energy intake and milk energy output. Suppression of the thyroid axis seems to be an essential aspect of the mechanism by which mice at 32.5 °C reduce their lactation performance to avoid overheating. However, THs do not define the upper limit to sustained energy intake and milk energy output at peak lactation at 21 °C. Another, as yet unknown, factor, prevents supplementary thyroxine exerting any stimulatory metabolic impacts on lactating mice at 21 °C.

Sex-specific behavioral and physiological changes during single parenting in a biparental species, Columba livia

Many species exhibit biparental care to maximize fitness. When a partner is lost, the surviving partner may alter their behavior to compensate offspring. Whether both sexes use the same physiological mechanisms to manifest their change in behavior remains elusive. We investigated behaviors and mechanisms associated with the alteration of parental care post-partner removal in a biparental avian species, the rock dove (Columba livia). We hypothesized that rock dove single parents experience sex-biased changes in neural genomic transcription and reproductive behaviors, and these changes are related to chick development. We manipulated parental partner presence and measured parental attendance, offspring growth, gene expression of glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) in the pituitary, and GR, MR, and estrogen receptor beta (ER-β) in the hypothalamus. We also measured circulating plasma concentrations of the stress-associated hormone corticosterone and the parental care-associated hormone prolactin. We also quantified prolactin gene (PRL) expression changes in the pituitary, as well as prolactin receptor (PRLR) expression in the hypothalamus and pituitary. We found that single mothers and fathers maintained similar provisioning levels as paired parents, but spent less cumulative time brooding chicks. Chicks of single parents were smaller than paired-parented chicks after three days post-hatch. Mothers in both treatment groups experienced higher expression of hypothalamic GR as compared to fathers. Single parents experienced lower PRL gene expression in the pituitary as compared to paired parents. No significant differences were found for the circulating hormones or other genes listed.

Transcriptome-based analysis of human peripheral blood reveals regulators of immune response in different viral infections.

There are difficulties in creating direct antiviral drugs for all viruses, including new, suddenly arising infections, such as COVID-19. Therefore, pathogenesis-directed therapy is often necessary to treat severe viral infections and comorbidities associated with them. Despite significant differences in the etiopathogenesis of viral diseases, in general, they are associated with significant dysfunction of the immune system. Study of common mechanisms of immune dysfunction caused by different viral infections can help develop novel therapeutic strategies to combat infections and associated comorbidities. To identify common mechanisms of immune functions disruption during infection by nine different viruses (cytomegalovirus, Ebstein-Barr virus, human T-cell leukemia virus type 1, Hepatitis B and C viruses, human immunodeficiency virus, Dengue virus, SARS-CoV, and SARS-CoV-2), we analyzed the corresponding transcription profiles from peripheral blood mononuclear cells (PBMC) using the originally developed pipeline that include transcriptome data collection, processing, normalization, analysis and search for master regulators of several viral infections. The ten datasets containing transcription data from patients infected by nine viruses and healthy people were obtained from Gene Expression Omnibus. The analysis of the data was performed by Genome Enhancer pipeline. We revealed common pathways, cellular processes, and master regulators for studied viral infections. We found that all nine viral infections cause immune activation, exhaustion, cell proliferation disruption, and increased susceptibility to apoptosis. Using network analysis, we identified PBMC receptors, representing proteins at the top of signaling pathways that may be responsible for the observed transcriptional changes and maintain the current functional state of cells. The identified relationships between some of them and virus-induced alteration of immune functions are new and have not been found earlier, e.g., receptors for autocrine motility factor, insulin, prolactin, angiotensin II, and immunoglobulin epsilon. Modulation of the identified receptors can be investigated as one of therapeutic strategies for the treatment of severe viral infections.

Structural Modeling of Cytokine-Receptor-JAK2 Signaling Complexes Using AlphaFold Multimer.

Homodimeric class 1 cytokine receptors include the erythropoietin (EPOR), thrombopoietin (TPOR), granulocyte colony-stimulating factor 3 (CSF3R), growth hormone (GHR), and prolactin receptors (PRLR). These cell-surface single-pass transmembrane (TM) glycoproteins regulate cell growth, proliferation, and differentiation and induce oncogenesis. An active TM signaling complex consists of a receptor homodimer, one or two ligands bound to the receptor extracellular domains, and two molecules of Janus Kinase 2 (JAK2) constitutively associated with the receptor intracellular domains. Although crystal structures of soluble extracellular domains with ligands have been obtained for all of the receptors except TPOR, little is known about the structure and dynamics of the complete TM complexes that activate the downstream JAK-STAT signaling pathway. Three-dimensional models of five human receptor complexes with cytokines and JAK2 were generated here by using AlphaFold Multimer. Given the large size of the complexes (from 3220 to 4074 residues), the modeling required a stepwise assembly from smaller parts, with selection and validation of the models through comparisons with published experimental data. The modeling of active and inactive complexes supports a general activation mechanism that involves ligand binding to a monomeric receptor followed by receptor dimerization and rotational movement of the receptor TM α-helices, causing proximity, dimerization, and activation of associated JAK2 subunits. The binding mode of two eltrombopag molecules to the TM α-helices of the active TPOR dimer was proposed. The models also help elucidate the molecular basis of oncogenic mutations that may involve a noncanonical activation route. Models equilibrated in explicit lipids of the plasma membrane are publicly available.

A novel signaling transduction pathway of melatonin on lactose synthesis in cows via melatonin receptor 1 (MT1) and prolactin receptor (PRLR).

In the current study, we explored the relationship between melatonin and lactose synthesis in in vivo and in vitro conditions. We found that long-term melatonin feeding to the dairy cows significantly reduced the milk lactose content in a dose dependent manner. This lactose reduction was not associated with a negative energy balance, since melatonin treatment did not alter the fat, glucose, or protein metabolisms of the cows. To identify the potential molecular mechanisms, the cow's mammary epithelial cells were cultured for gene expression analysis. The results showed that the effect of melatonin on lactose reduction was mediated by its receptor MT1. MT1 activation downregulated the mRNA expression of the prolactin receptor gene (PRLR), which then suppressed the gene expression of SLC35B1. SLC35B1 is a galactose transporter and is responsible for the transportation of galactose to Golgi apparatus for lactose synthesis. Its suppression reduced the lactose synthesis and the milk lactose content. The discovery of this signal transduction pathway of melatonin on lactose synthesis provides a novel aspect of melatonin's effect on carbohydrate metabolism in cows and maybe also in other mammals, including humans.

Impaired microRNA-21 and prolactin receptor levels in the decidua of the prepubertal offspring of diabetic rats are prevented by maternal diets enriched in olive oil

Impaired microRNA-21 and prolactin receptor levels in the decidua of the prepubertal offspring of diabetic rats are prevented by maternal diets enriched in olive oil

The role of prolactin in the suppression of the response to restraint stress in the lactating mouse.

Suppression of the hypothalamic-pituitary-adrenal (HPA) axis is a well-characterised maternal adaptation that limits the exposure of the offspring to maternally-derived stress hormones. This current study has investigated the possible involvement of the lactogenic hormone, prolactin, in this physiologically important adaptation. As expected, circulating prolactin levels were higher in unstressed lactating mice compared to their virgin counterparts. Interestingly however, the ability of an acute period of restraint stress to further elevate prolactin levels was diminished in the former group. The stress-induced rise in prolactin levels in the virgin animals was concurrent with an increase in prolactin receptor activation within the adrenal cortical cells. This adrenal response was not seen in either the stressed or control lactation group, an observation that may be in part explained by the observed downregulation of prolactin receptor mRNA expression within this tissue. Further evidence of suppression of the HPA axis during lactation was revealed using in situ hybridisation to demonstrate that while acute restraint stress increased corticotrophin releasing hormone (CRH) mRNA expression in the hypothalamic paraventricular nucleus in both virgin and lactating mice, the magnitude of this response was reduced in the latter group. This potentially adaptive response did not, however, appear to result from the altered prolactin profile during lactation because it was not affected by the pharmacological suppression of prolactin secretion from the pituitary. This study therefore suggests that during lactation the response of the HPA axis to stress is suppressed at multiple physiological levels which are mediated by both prolactin-dependent and prolactin-independent mechanisms.

Developmental and reproductive performance differences of the slick-hair Holstein

Heat stress causes detrimental effects on animals, such as dairy cows and heifers, resulting in decreased production and changes in physiological aspects. Some Holsteins carry a mutation in the prolactin receptor (Slick; SL) that causes them to have shorter hair and greater heat tolerance compared with the wild-type (WT) cattle. Slick animals have lower temperatures, greater milk production and shorter calving intervals. The objectives of the current study were to describe monthly weight, reproductive performance, and mortality rates of the SL Holstein compared with the WT. Experiments were conducted at the Agricultural Experiment Substation in Gurabo, Puerto Rico, to determine average monthly weights of 88 heifers (40 SL and 48 WT) born between 2017 and 2020. Heifers’ estrus (n=22 SL; n=27 WT) was synchronized followed by timed artificial insemination to determine differences in pregnancy rates. A subset of lactating cows was used to compare number of services required for pregnancy. Mortality frequency between genotypes was analyzed and compared. Data were analyzed using SAS University Edition, 2018 Proc-GLIMMIX and Tukey test. Compared to WT heifers, SL heifers were heavier after 14 months, required one to two fewer insemination services and died at a lower frequency rate (22% vs. 78%). Genetically selecting heat tolerant SL cows over WT may improve reproductive performance.

The Diversity of Gut Microbiota at Weaning Is Altered in Prolactin Receptor-Null Mice.

Maternal milk supports offspring development by providing microbiota, macronutrients, micronutrients, immune factors, and hormones. The hormone prolactin (PRL) is an important milk component with protective effects against metabolic diseases. Because maternal milk regulates microbiota composition and adequate microbiota protect against the development of metabolic diseases, we aimed to investigate whether PRL/PRL receptor signaling regulates gut microbiota composition in newborn mice at weaning. 16SrRNA sequencing of feces and bioinformatics analysis was performed to evaluate gut microbiota in PRL receptor-null mice (Prlr-KO) at weaning (postnatal day 21). The normalized colon and cecal weights were higher and lower, respectively, in the Prlr-KO mice relative to the wild-type mice (Prlr-WT). Relative abundances (Simpson Evenness Index), phylogenetic diversity, and bacterial concentrations were lower in the Prlr-KO mice. Eleven bacteria species out of 470 differed between the Prlr-KO and Prlr-WT mice, with two genera (Anaerotruncus and Lachnospiraceae) related to metabolic disease development being the most common in the Prlr-KO mice. A higher metabolism of terpenoids and polyketides was predicted in the Prlr-KO mice compared to the Prlr-WT mice, and these metabolites had antimicrobial properties and were present in microbe-associated pathogenicity. We concluded that the absence of the PRL receptor altered gut microbiota, resulting in lower abundance and richness, which could contribute to metabolic disease development.

A novel LncRNA SPIRE1/miR-181a-5p/PRLR axis in mandibular bone marrow-derived mesenchymal stem cells regulates the Th17/Treg immune balance through the JAK/STAT3 pathway in periodontitis.

Periodontitis is a microbial-related chronic inflammatory disease associated with imbalanced differentiation of Th17 cells and Treg cells. Bone marrow-derived mesenchymal stem cells (BM-MSCs) possess wide immunoregulatory properties. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) contribute to the immunomodulation in the pathological mechanisms of inflammatory diseases. However, critical lncRNAs/miRNAs involved in immunomodulation of mandibular BM-MSCs largely remain to be identified. Here, we explored the molecular mechanisms behind the defective immunomodulatory ability of mandibular BM-MSCs under the periodontitis settings. We found that mandibular BM-MSCs from P. gingivalis-induced periodontitis mice had significantly reduced expression of LncRNA SPIRE1 than that from normal control mice. LncRNA SPIRE1 knockdown in normal BM-MSCs caused Th17/Treg cell differentiation imbalance during the coculturing of BM-MSCs and CD4 T cells. In addition, LncRNA SPIRE1 was identified as a competitive endogenous RNA that sponges miR-181a-5p in BM-MSCs. Moreover, miR-181a-5p inhibition attenuated the impact of LncRNA SPIRE1 knockdown on the ability of BM-MSCs in modulating Th17/Treg balance. Prolactin receptor (PRLR) was validated as a downstream target of miR-181a-5p. Notably, targeted knockdown of LncRNA SPIRE1 or PRLR or transfection of miR-181a-5p mimics activated the JAK/STAT3 signaling in normal BM-MSCs, while treatment with STAT3 inhibitor C188-9 restored the immunomodulatory properties of periodontitis-associated BM-MSCs. Furthermore, BM-MSCs with miR-181a-5p inhibition or PRLR-overexpression showed enhanced in vivo immunosuppressive properties in the periodontitis mouse model. Our results indicate that the JAK/STAT3 pathway is involved in the immunoregulation of BM-MSCs, and provide critical insights into the development of novel targeted therapies against periodontitis.

Jiawei Shoutai Pill promotes decidualization by regulating the SGK1/ENaC pathway in recurrent spontaneous abortion

Ethnopharmacological relevanceJiawei Shoutai Pill (JWSTW) is a traditional herbal formula for recurrent spontaneous abortion (RSA). Although JWSTW significantly improves the clinical symptoms of RSA patients, its molecular mechanism remains unclear. Aim of studyThis study evaluated the expression and function of the serum/glucocorticoid regulated kinase 1/epithelial sodium channel (SGK1/ENaC) pathway and decidualization level in RSA patients and mice. It also investigated the therapeutic effects and potential mechanisms of JWSTW. Materials and methods30 early RSA patients and 30 normal pregnant women undergoing induced abortion during the same period were included in the study. Decidual tissues were collected, and HE staining, immunohistochemistry, Western blot, and RT-PCR were used to detect protein and mRNA expression levels of SGK1, ENaC-a, estrogen Rreceptor β (ERβ), and progesterone receptor (PR) in patients’ decidual tissues. Protein expression levels of prolactin receptor (PRLR) and insulin-like growth factor binding protein 1 (IGFBP-1) were also detected. A classical RSA mouse model was constructed, and the mice were randomly divided into four groups: normal, model, dydrogesterone (DQYT) (0.33 g/kg/d), and JWSTW (1.66 g/kg/d). The normal and model groups received the same volume of distilled water by gavage for 8 and 14 days after pregnancy. On the 14th day of pregnancy, the embryonic loss rate of each group, the number of offspring born to naturally delivered mice, and the protein or mRNA expression levels of key factors of the SGK1/ENaC pathway (SGK1, ENaC-a, ERβ, and PR), decidual proliferation marker (Ki67), mesenchymal-epithelial transition (E-cadherin and Vimentin), and decidualization markers (PRLR and IGFBP-1) in mouse decidual tissue on the eighth day of pregnancy were observed. ResultsThe decidual tissue structure of RSA patients was abnormal. Immunohistochemical analysis revealed significantly reduced positive expression of SGK1, ENaC-a, ERβ, and PR proteins in the decidual tissue of RSA patients (P < 0.001). Western blot and RT-PCR analyses demonstrated significantly decreased protein and mRNA expression of SGK1, ENaC-a, ERβ, and PR in the decidual tissue of RSA patients (all P < 0.05). Additionally, protein expression of PRLR and IGFBP-1 was significantly reduced (both P < 0.001). The RSA mouse model exhibited a significant increase in embryo loss rate and decreased litter size (both P < 0.001). Treatment with DQYT and JWSTW rescued the embryo loss rate and litter size to varying extents (all P < 0.05). The protein or mRNA expression levels of SGK1, ENaC-a, ERβ, PR, Ki67, E-cadherin, vimentin, PRLR, and IGFBP-1 in RSA mice were improved to different degrees after treatment with DQYT and JWSTW (all P < 0.05). ConclusionsAbnormal SGK1/ENaC signaling pathway regulation is closely associated with early endometrial damage in RSA patients. JWSTW promotes endometrial proliferation and mesenchymal-epithelial transition through the SGK1/ENaC signaling pathway, improving endometrial shedding. Consequently, JWSTW is a potential treatment for RSA.

Cancer stem cells as the source of tumor associated myoepithelial cells in the tumor microenvironment developing ductal carcinoma in situ

The heterogeneous cell population in the stromal microenvironment is considered to be attributed to the multiple sources from which the cells originate. Tumor associated myoepithelial cells (TAMEs) are one of the most important populations in the tumor microenvironment (TME) especially in breast cancer. On the other hand, cancer stem cells (CSCs) have previously been described to be the origin of tumor-associated cellular components in the TME. We prepared a cancer stem cell model converting mouse-induced pluripotent stem cells (miPSCs) in the presence of conditioned medium of breast cancer cell line MDA-MB-231 cells. The converted cells developed tumors progressing into invasive carcinoma with ductal carcinoma in situ (DCIS) like structure when transplanted into mouse mammary fat pads. The primary cultured cells from the tumor further exhibited markers of CSC such as Sox2, Oct3/4, - CD133 and EpCAM, and mammary gland-related TAME markers such as α-smooth muscle actin, cytokeratin 8, whey acidic protein, prolactin receptor and progesterone receptor as well. These results indicated that the CSCs could be an origin of TAMEs contributing to mammary gland epithelial cell differentiation and the progression to invasive carcinoma during tumor development. The gene expression profiles confirmed the enhanced signaling pathways of PI3K/AKT and MAPK, which have been demonstrated to be enriched in the CSC models, together with the estrogen receptor signaling which was peculiar to mammary gland-derived character.

Structure and function of a dual antagonist of the human growth hormone and prolactin receptors with site-specific PEG conjugates

Human growth hormone (hGH) is a pituitary derived endocrine protein that regulates several critical postnatal physiologic processes including growth, organ development, and metabolism. Following adulthood, GH is also a regulator of multiple pathologies like fibrosis, cancer, and diabetes. Therefore, there is a significant pharmaceutical interest in developing antagonists of hGH action. Currently there is a single FDA approved antagonist of the hGH receptor (hGHR) prescribed for treating patients with acromegaly and discovered in our laboratory almost three decades ago. Here, we present the first data on the structure and function of a new set of protein antagonists with the full range of hGH actions - dual antagonists of hGH binding to the GHR as well as that of hGH binding to the prolactin receptor. We describe the site-specific poly-ethylene-glycol (PEG) conjugation, purification, and subsequent characterization using MALDI-TOF, size-exclusion chromatography, thermostability, and biochemical activity in terms of ELISA-based binding affinities with GHR and PRLR. Moreover, these novel hGHR antagonists display distinct antagonism of GH induced GHR intracellular signaling in vitro and marked reduction in hepatic insulin-like growth factor 1 output in vivo. Lastly, we observed potent anti-cancer biological efficacies of these novel hGHR antagonists against human cancer cell lines. In conclusion, we propose that these new GHR antagonists have potential for development towards multiple clinical applications related to GH associated pathologies.

Comparative Study on Umbilical Cord Serum Prolactin Levels of Normal Preterm Neonates and Preterm Neonates with Respiratory Distress Syndrome

Introduction: Respiratory distress syndrome (RDS) is the most common cause of respiratory distress, morbidity, and mortality associated with premature delivery. Prolactin is present in very high concentrations in amniotic fluid, and prolactin receptors are present in lung preparations. Prolactin acts as a direct trigger of lecithin synthesis or in a “chain reaction” along the hypothalamic–pituitary–adrenal axis. Thus, prolactin seems to have a role in lung maturation along with many other factors through a complex mechanism. Aim: The aim of this study was to evaluate and compare the umbilical cord serum prolactin levels of preterm infants with and without RDS. Materials and Methods: This was a hospital-based comparative observational study carried out at neonatal units of the department of pediatric medicine in a tertiary care center from April 2019 to May 2020. Out of a total of 80 preterm infants, 40 developed RDS and other 40 served as controls. Umbilical cord blood prolactin levels in neonates were measured in both groups and compared. Chi-square test and an unpaired Student’s t-test were used for statistical analysis. Probability was considered significant if &lt;0.05. Results: The mean weight of babies was 1312.50 ± 134.30 g in the normal preterm group and 1285.80 ± 314.76 g in the preterm RDS group (P = 0.622). The mean gestational age was 32.1 ± 1.39 weeks in the normal preterm group and 31.77 ± 2.10 weeks in the preterm RDS group (P = 0.418). The mean serum prolactin levels were slightly high in the female preterm RDS group (P = 0.533). The mean cord prolactin levels in all the gestational ages were higher in normal preterm as compared to preterm with RDS (P &lt; 0.05). Conclusions: Prolactin level was significantly associated with gestational age and birth weight. Low prolactin levels were significantly associated with the development of RDS in preterm babies. The easily available cord blood can be used to measure prolactin levels at birth to predict RDS.