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Placensin is a glucogenic hormone secreted by human placenta - PubMed

  • ️Wed Jan 01 2020

. 2020 Jun 4;21(6):e49530.

doi: 10.15252/embr.201949530. Epub 2020 Apr 24.

Affiliations

Placensin is a glucogenic hormone secreted by human placenta

Yiping Yu et al. EMBO Rep. 2020.

Abstract

FBN1 encodes asprosin, a glucogenic hormone, following furin cleavage of the C-terminus of profibrillin 1. Based on evolutionary conservation between FBN1 and FBN2, together with conserved furin cleavage sites, we identified a peptide hormone placensin encoded by FBN2 based on its high expression in trophoblasts of human placenta. In primary and immortalized murine hepatocytes, placensin stimulates cAMP production, protein kinase A (PKA) activity, and glucose secretion, accompanied by increased expression of gluconeogenesis enzymes. In situ perfusion of liver and in vivo injection with placensin also stimulate glucose secretion. Placensin is secreted by immortalized human trophoblastic HTR-8/SVneo cells, whereas placensin treatment stimulates cAMP-PKA signaling in these cells, accompanied by increases in MMP9 transcripts and activities, thereby promoting cell invasion. In pregnant women, levels of serum placensin increase in a stage-dependent manner. During third trimester, serum placensin levels of patients with gestational diabetes mellitus are increased to a bigger extent compared to healthy pregnant women. Thus, placensin represents a placenta-derived hormone, capable of stimulating glucose secretion and trophoblast invasion.

Keywords: gluconeogenesis; hormone; invasiveness; placensin.

© 2020 The Authors.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Prediction of a peptide hormone in the C‐terminal region of FBN2

  1. Exon–intron structures for human FBN1 and FBN2 genes. Conserved furin cut sites are indicated. Asprosin (Asp) is encoded by exons 65–66 of the FBN1 gene, whereas a conserved Asp‐like sequence is found in exons 64–65 of FBN2.

  2. Sequence comparison between C‐terminal regions of human FBN1 and FBN2, encoding asprosin, and placensin, respectively. Asterisks indicate identical residues.

  3. Conservation of FBN2 furin cleavage sites and C‐terminal regions among diverse vertebrate species. Boxed area represents conserved furin cleavage site. Asterisks indicate identical residues conserved in all species examined, colons indicate highly conserved residues.

Figure EV1
Figure EV1. Placensin sequence comparison and tissue distribution, together with asprosin serum levels, FBN2 expression, and placensin‐furin co‐localization
  1. A

    Conservation of furin cleavage sites and placensin sequences in diverse mammals. Boxed area represents conserved furin cleavage site. Asterisks indicate identical residues conserved in all species examined, colons indicate highly conserved residues, dots indicate partly conserved residues.

  2. B, C

    FBN2 transcripts and proteins are expressed mainly in human placenta (data from BioGPS and Human Protein Atlas, respectively).

  3. D

    Serum asprosin levels during first, second, and third trimesters (biological replicates, n = 10 in each group).

  4. E

    FBN2 expression is much higher in cytotrophoblasts (CTB) than syncytiotrophoblasts (STB) and stromal cells (STR) in human placenta at 8 weeks of pregnancy based on single‐cell RNA‐seq analyses. Data (in violin plot) were derived from GEO accession number GSE89497 at NCBI [24, Data ref: 25]. The white dot represents the median; the black bar in the center of violin indicates the interquartile range (between first and third quartile); the thin black line cross the bar indicates the lower first quartile and the upper third quartile.

  5. F

    Placensin and furin co‐localization using immunostaining. Scale bar: 20 μm.

Data information: Bars are shown as mean ± SEM. (D) **P < 0.01, ANOVA followed by Tukey's multiple comparison tests was used for comparison.Source data are available online for this figure.

Figure 2
Figure 2. High expression of FBN2 and placensin in human, but not mouse, placenta

  1. FBN2 mRNA levels in multiple human tissues. Human tissue cDNAs were used as templates for PCR of FBN2, followed by agarose gel analyses. GAPDH levels served as loading controls (biological replicates, n = 2).

  2. FBN2 mRNA levels in mouse tissues based on quantitative RT–PCR. Data were normalized based on levels in adipose tissues.

  3. Comparison of FBN2 transcript levels between human and mouse placentas. Common primers for FBN2 and GAPDH in both species were used for RT–PCR analyses (biological replicates, n = 3).

  4. Immuno‐histochemical detection of placensin in human placental villi obtained from women at 9 and 38 weeks of pregnancy. During early pregnancy, placensin immunostaining was found in cytotrophoblasts (c), whereas syncytiotrophoblasts (s) showed weak staining. At 38 weeks of pregnancy, strong placensin signals were detected in both syncytiotrophoblasts and cytotrophoblasts (arrows). Staining using E‐cadherin antibodies confirmed the identity of cytotrophoblasts (inset showing higher magnification E‐cadherin (E‐Cad) staining). Lower panels depict sections stained with nonimmune IgG as negative controls. Scale bars, 100 μm.

  5. Immunoblotting of placensin in human placental villi (term pregnancy). Samples from three individual patients were processed with or without N‐glycosidase pre‐treatment to reveal untreated and deglycosylated placensin.

  6. For prokaryotic cell expression, a cDNA fragment corresponding to residues 2,779–2,912 of the human FBN2 coding region was appended with GST and 6‐histidine tags and subcloned into the PGEX‐6P‐1 vector for expression in Escherichia coli. Left panel: Coomassie Blue staining of proteins in the SDS–PAGE gel before and after digestion with the PreScission protease. 1: Flow‐through; 2: washes; 3: before PreScission treatment; 4: after PreScission treatment. Right panel: Immunoblotting using placensin antibodies.

  7. Immunoblotting of recombinant placensin generated in E. coli and CHO cells. For CHO cell expression, placensin cDNA sequence was subcloned into an adenoviral vector pAV[Exp]‐CMV downstream of the IgK signal peptide under the control of the CMV promoter for infection of cells before immunoblotting of media 2 days later. Recombinant placensin proteins secreted from CHO cells (b) showed higher molecular weight than those from bacteria (a). Following treatment with N‐glycosidase to remove N‐linked carbohydrate side chains (c), prokaryotic‐ and eukaryotic cell‐derived placensin proteins showed similar sizes.

Data information: Bars are shown as mean ± SEM. For C: a,b, groups with different letters were significantly different (P < 0.05) by parametric unpaired Welch's t‐test.

Figure 3
Figure 3. Placensin stimulation of cAMP production, PKA activity, and glucose release in hepatocytes and promotion of glucose secretion in vivo

  1. Prokaryotic cell‐derived placensin stimulated cAMP production (at 10 min), and PKA activity (at 30 min) by primary mouse hepatocytes and hepatocyte‐derived AML12 cells (biological replicates, n = 5).

  2. Placensin stimulation of glucose secretion (at 5 h) and key gluconeogenesis gene transcripts (at 1 h) in both cell types (biological replicates, n = 4). Transcript levels for PEPCK and G6Pase were determined using quantitative RT–PCR. Treatment with glucagon (10 nM) served as positive controls.

  3. Liver from adult female mice was perfused in situ with placensin (1.0 μg/pulse) or PBS before measurement of hepatic glucose output from inferior vena cava (biological replicates, n = 3).

  4. Adult female mice were injected intraperitoneally with placensin (30 μg/injection) with blood sampling at different time points for glucose levels (biological replicates, n = 5).

Data information: Bars are shown as mean ± SEM. For A and B, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by ANOVA followed by Brown–Forsythe and Welch's test; for C, *P < 0.05, ****P < 0.0001, significant differences between placensin injection groups and corresponding control groups by parametric unpaired Welch's t‐test; for D, **P < 0.01, significant differences between placensin injection groups and corresponding control groups by nonparametric Mann–Whitney t‐test, and *P < 0.05 and ****P < 0.0001, significant differences between control groups and placensin injection groups by parametric unpaired Welch's t‐test.Source data are available online for this figure.

Figure EV2
Figure EV2. Asprosin, placensin, glucagon, and insulin actions in diverse cells

  1. Asprosin stimulation of cAMP and glucose secretion by mouse primary hepatocytes (biological replicates, cAMP: n = 4; glucose: n = 6).

  2. Interactions between glucagon/insulin and placensin on glucose secretion in primary hepatocytes (biological replicates, placensin: n = 6, glucagon: n = 3, insulin: n = 3, glucagon/insulin + placensin: n = 3).

  3. Lack of placensin stimulation of cAMP by intestinal IEC‐6 (biological replicates, n = 4) and kidney MDCK cells (biological replicates, n = 3).

Data information: Bars are shown as mean ± SEM. ANOVA followed by Brown–Forsythe and Welch's test was used for comparison. When comparing with control groups, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.Source data are available online for this figure.

Figure 4
Figure 4. Placensin secretion and promotion of trophoblastic cell cAMP production, PKA activity, MMP9 expression, and invasion

  1. Left panel: Immunoblotting of placensin content in HTR‐8/SVneo, but not BeWo, JAR, and JEG3, cells. Right panel: Time‐dependent secretion of placensin by HTR‐8/SVneo, but not JAR, cells monitored using an ELISA (biological replicates, n = 4–5).

  2. Knockdown of FBN2 in HTR‐8/SVneo cells led to decreases in FBN2 transcripts and placensin secretion. Cells were treated with FBN2 siRNA (SiFBN2; 20 nM) or control siRNA (SiC; 20 nM) for 6 h. Following media change, cells and conditioned media were collected at 48 h after incubation. Placensin transcripts and proteins were determined by RT–PCR and ELISA (biological replicates, n = 8), respectively. Lower panel: immunoblotting of secreted placensin by HTR‐8/SVneo.

  3. Placensin stimulation of cAMP production and PKA activities by HTR‐8/SVneo cells. Cells were treated with placensin for 10 min before cAMP measurement (biological replicates, n = 5). For PKA activities, cells were treated with placensin (50 ng/ml) for 30 min. Treatment with forskolin (10 μM) served as positive controls.

  4. Placensin stimulation of transcript levels for MMP9 but not MMP2 (biological replicates, n = 12). Cells were treated with placensin for 24 h before RT–PCR analyses of transcript levels.

  5. Placensin stimulation of MMP9, but not MMP2, activities without affecting cell proliferation. After 24 h of treatment with placensin, media were collected for zymographic and densitometric analyses as well as determination of total cell numbers (biological replicates, n = 4).

  6. Placensin stimulation of HTR‐8/SVneo cell invasion. After placensin treatment for 24 h, cell invasiveness was determined using the Transwell assay (left panel: micrographs of cells; right panel: fold changes in number of migrated cells, biological replicates, n = 4). Cells were incubated with mitomycin C to rule out effects on cell proliferation. Scale bars: 100 μm. C: control.

Data information: Bars are shown as mean ± SEM. For B, ANOVA followed by Tukey's multiple comparison tests was used for comparison. For C, ANOVA followed by Brown–Forsythe and Welch's test was used for comparison. For D–F, parametric unpaired Welch's t‐test was used. When comparing with control groups, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.Source data are available online for this figure.

Figure EV3
Figure EV3. Mass spec of placental cell media and modulation of placental cell functions by asprosin and placensin

  1. Mass spec of HTR‐8/SVneo conditioned media. HTR‐8/SVneo conditioned media was precipitated using placensin antibody before mass spec analyses. Fragments detected were underlined and highlighted using different colors.

  2. Mass spec of sera from women at term pregnancy. Sera from women at term pregnancy were immunoprecipitated using placensin antibody before mass spec analyses. Fragments detected were underlined and highlighted using different colors.

  3. Asprosin stimulation of invasiveness of cultured HTR‐8/SVneo cells (biological replicates, n = 3).

  4. Placensin stimulation of cAMP production by BeWo, JEG‐3, but not JAR, cells (biological replicates, BeWo: n = 3, JEG‐3: n = 5, JAR:n = 3).

Data information: Bars are shown as mean ± SEM. ANOVA followed by Brown–Forsythe and Welch's test was used for comparison. When comparing with control groups, *P < 0.05, **P < 0.01, and ****P < 0.0001.Source data are available online for this figure.

Figure 5
Figure 5. Increases in serum placensin and minimal changes in glucagon levels during human pregnancy and in patients with gestational diabetes mellitus (GDM)

  1. Immunoblotting of serum placensin from patients at second trimester (1–3), term pregnancy (4–6) using specific placensin antibodies.

  2. Serum levels of placensin and glucagon at different weeks of pregnancy measured using specific ELISA in normal and GDM patients. Numbers in parentheses represent number of patients.

  3. Ages of patients at different gestational stages and BMI values for normal pregnant women and GDM patients.

Data information: Bars are shown as mean ± SEM. *P < 0.05, nonparametric Mann–Whitney's t‐test was used for comparison between GDM groups with corresponding control groups.Source data are available online for this figure.

Figure EV4
Figure EV4. Immunoblotting of serum placensin in both normal and GDM term pregnancies showing a band around 20 kDa
Figure EV5
Figure EV5. Characteristics of placensin ELISA

  1. Hormonal specificity of placensin ELISA. Recombinant asprosin and hCG showed negligible cross‐reactivity.

  2. Serum samples (1 and 2) showed parallel reactivity curves similar to recombinant placensin in the placensin ELISA.

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