Teh-Yuan Ho

Milk-borne lactocrine-acting factors affect gene expression patterns in the developing neonatal porcine uterus

Lactocrine communication of milk-borne bioactive factors (MbFs) from mother to offspring through nursing can affect neonatal development with lasting consequences. Relaxin (RLX), a lactocrine-active peptide found in porcine colostrum, stimulates estrogen receptor-α (ESR1) expression required for uterine development shortly after birth (postnatal day=PND 0). Whether other MbFs or cooperative lactocrine mechanisms affect the neonatal uterine developmental program is unknown. To determine the effects of age, nursing, and exogenous RLX on gene expression associated with uterine development, gilts (n=4-5/group) were assigned to nurse ad libitum or to receive milk replacer, with or without exogenous RLX (20 μg/kg BW i.m./6 h for 48 h), from birth to PND 2 when uteri were collected. Body weight and uterine weight increased (P<0.05) similarly from birth to PND 2 in all gilts. However, colostrum consumption was required for normal uterine ESR1, vascular endothelial growth factor (VEGFA), matrix metalloproteinase 9 (MMP9), and RLX receptor (RXFP1) protein and/or transcript expression on PND 2. Uterine ESR1, VEGFA, and MMP9 protein levels were below (P<0.01) the assay sensitivity in replacer-fed gilts. Supplemental RLX increased (P<0.05) uterine ESR1 protein and mRNA in nursed gilts, as well as VEGFA protein in nursed and VEGFA mRNA in both nursed and replacer-fed gilts. RLX treatment did not affect uterine MMP9 mRNA levels. When compared with replacer-fed gilts on PND 2, uterine RXFP1 mRNA was reduced (P<0.05) in nursed gilts and in RLX-supplemented replacer-fed gilts. These results constitute the first evidence that establishment of the neonatal porcine uterine developmental program requires maternal lactocrine support.

Relaxin-induced matrix metalloproteinase-9 expression is associated with activation of the NF-κB pathway in human THP-1 cells

Matrix metalloproteinases (MMPs) and relaxin (RLX) are reported to play an important role in tissue remodeling and wound repair. When macrophages populate wound sites, they secrete biologically active substances, including MMPs. The transcription factor NF‐κB is important in MMP gene regulation in macrophage cells. Thus, a monocyte/macrophage cell line, THP‐1, was used to study the molecular mechanism of RLX action on MMP‐2 and MMP‐9 expression. After 24 h incubation with porcine RLX (100 ng/ml), conditioned media (CM) and THP‐1 cells were collected. Gelatin zymography demonstrated an increase in pro‐MMP‐9 activity in response to RLX in CM, and no significant change in pro‐MMP‐2 expression was observed. Immunoblot analysis also revealed an increase in pro‐MMP‐9 in CM from RX‐treated THP‐1 cells. Gel EMSA showed that NF‐κB DNA‐binding activity was elevated in THP‐1 cells treated with RLX for 10 min and reached a peak at 30 min. The NF‐κB DNA complex was supershifted using antibodies against NF‐κB subunits p50 and p65. Increased expression of the p50 and p65 NF‐κB subunits was also detected in THP‐1 cells after RLX treatment. Incubation with RLX (90 min) reduced THP‐1 expression of the NF‐κB inhibitor protein, IκB‐α. Using a specific NF‐κB inhibitor, pyrrolidine dithiocarmate (PDTC) inhibited nuclear binding of NF‐κB. Pre‐exposure to PDTC suppressed pro‐MMP‐9 activity and protein levels in RLX‐treated THP‐1 cells. In conclusion, these data suggest that RLX‐induced tissue remodeling through increasing MMP‐9 expression is dependent on NF‐κB activation.

Effects of relaxin and estrogens on bone remodeling markers, receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG), in rat adjuvant-induced arthritis

Rheumatoid arthritis (RA) is characterized by joint inflammation and bone destruction. The receptor activator of nuclear factor-kappa B ligand (RANKL)-osteoprotegerin (OPG) system is important for maintaining the balance between bone resorption and formation. Both serum RANKL/OPG protein and synovial tissue RANKL/OPG mRNA ratios are elevated in patients with RA. Studies indicate that hormones of pregnancy, estrogens and relaxin, administered in combination, reduce circulating (TNF)-α and joint inflammation in a rat adjuvant-induced arthritis (AIA) model of RA. The purpose of this study was to investigate whether relaxin (RLX), alone or in combination with estrogens, regulates the bone remodeling markers RANKL and OPG in vivo and in vitro. Results show that in AIA rats, treatment with relaxin, estradiol valerate (EV) or EV in combination with relaxin had no effect on circulating RANKL. However, EV increased systemic OPG and combined treatment with EV and relaxin further increased circulating OPG in comparison to EV alone. Importantly, the RANKL/OPG protein ratio was lower in rats treated with EV or EV+RLX when compared to arthritic controls. Relaxin in combination with EV decreased local RANKL transcripts, increased OPG mRNA and decreased the RANKL/OPG mRNA ratio in joints of arthritic rats when compared to controls. RLX family peptide receptor 1 (RXFP1) gene expression in joints of AIA rats increased in response to EV and EV+RLX. In parathyroid hormone-pretreated murine UMR 106-01 osteoblast cells, 17-β-estradiol (E) and E+RLX increased RXFP1 transcripts, while RLX reduced RANKL mRNA and protein expression. However, in vitamin D-treated primary rat osteoblast cells E+RLX increased OPG protein and reduced the RANKL/OPG protein ratio. These results suggest that modulation of the RANKL-OPG system by estrogens and RLX may contribute to their antiarthritic effects on bone during pregnancy.

Transient estrogen exposure from birth affects uterine expression of developmental markers in neonatal gilts with lasting consequences in pregnant adults.

Disruption of estrogen-sensitive, estrogen receptor (ER)-dependent events during porcine uterine development between birth (postnatal day=PND 0) and PND 14 affects patterns of uterine morphoregulatory gene expression in the neonate with lasting consequences for reproductive success. Uterine capacity for conceptus support is reduced in pregnant adult gilts exposed to estradiol valerate (EV) for 14 days from birth. Objectives here were to determine effects of EV exposure from birth through PND 13 on neonatal uterine and adult endometrial markers of growth, patterning, and remodeling. Targets included the relaxin receptor (RXFP1), estrogen receptor-alpha (ESR1) and vascular endothelial growth factor (VEGFA), morphoregulatory markers HOXA10 and WNT7A, and the matrix metalloproteinases (MMP)2 and MMP9. Gilts were treated daily with EV (50 microg/kg body weight per day, i.m.) or corn oil vehicle from birth through PND 13. Uteri were obtained from neonates on PND 14 and from adults on pregnancy day 12 (PxD 12). In neonates, EV exposure from birth increased uterine RXFP1 gene expression, and both ESR1 and VEGFA proteins. At PxD 12, endometrial RXFP1 mRNA remained elevated, while ESR1 protein was reduced. Early EV treatment decreased neonatal uterine WNT7A, but increased HOXA10 expression. WNT7A expression was reduced in EV-treated adults. Transient EV exposure increased MMP9 transcripts at PND 14, whereas both latent and active MMP9 activity was increased due to early EV treatment in adults on PxD 12. Results support the hypothesis that transient, estrogen-induced disruption of porcine uterine development from birth alters early programming events that lead to functional consequences in the adult.

Characterization and biological activity of relaxin in porcine milk.

A lactocrine mechanism for delivery of maternally derived relaxin (RLX) into the neonatal circulation as a consequence of nursing was proposed for the pig. Immunoreactive RLX was detected in colostrum and in the serum of newborn pigs only if they were allowed to nurse. Milk-borne RLX concentrations are highest during early lactation (9-19  ng/ml), declining to <2  ng/ml by postnatal day 14. Whether milk-borne RLX is bioactive is unknown. Evidence that RLX concentrations in milk are higher than in maternal circulation in several species suggests the mammary gland as a site of local RLX production. It is unknown whether the porcine mammary gland is a source of RLX. Therefore, objectives were to evaluate RLX bioactivity in porcine milk during the first 2 weeks of lactation, identify the form of RLX in porcine milk, and determine whether mammary tissue from early lactation is a source of milk-borne RLX. Milk RLX bioactivity was determined using an in vitro bioassay in which cAMP production by human embryonic kidney (HEK293T) cells transfected with the human RLX receptor (RXFP1) was measured. RLX bioactivity was highest at lactation day (LD) 0, decreasing to undetectable levels by LD 4. Immunoblot analysis of milk proteins revealed an 18  kDa band, indicating proRLX as the primary form of RLX in porcine milk. ProRLX protein and transcripts were detected in porcine mammary tissue on LD 0 and 7. Results support the lactocrine hypothesis by defining the nature and a potential source for bioactive proRLX in porcine colostrum/milk.

Relaxin Induces Matrix Metalloproteinase-9 through Activation of Nuclear Factor Kappa B in Human THP-1 Cells

Abstract: Matrix metalloproteinase (MMP) and relaxin are important for tissue remodeling and wound repair. Macrophages populate wound sites and secrete MMPs. Nuclear factor kappa B (NF‐κB) is linked to MMP gene regulation. Thus, a monocyte/macrophage cell line, THP‐1, was used to study the mechanism of relaxins action on MMPs. Relaxin increased MMP‐9 protein and activity in THP‐1 cell‐conditioned media, with no significant change in MMP‐2 activity. NF‐κB DNA binding activity was elevated in response to relaxin, and supershift assay showed activation of both NF‐κB subunits p50 and p65. Relaxin also reduced NF‐κB inhibitor protein, IκB‐α. In conclusion, these data suggest that relaxin‐induced MMP‐9 expression in THP‐1 cells involves NF‐κB activation.

Biological activity of relaxin in porcine milk.

A lactocrine mechanism for delivery of maternally derived relaxin (RLX) into the neonatal circulation as a consequence of nursing has been proposed for the pig. Consistently, immunoreactive porcine RLX was detected in colostrum as well as in the serum of nursing pigs. Concentrations of porcine RLX in milk are highest during early lactation (9–19 ng/mL) and decline to less than 2 ng/mL by postnatal day 14. However, RLX bioactivity has not been described in porcine milk. Therefore, this study was designed to establish an assay for RLX bioactivity in porcine milk and to determine if milk RLX bioactivity was related to RLX concentrations in milk collected at parturition (lactation day 0) and on lactation day 14. To assess milk RLX bioactivity, an in vitro bioassay using human embryonic kidney (HEK293T) cells transfected with the human RLX receptor (LGR7) was developed. Milk RLX bioactivity was confirmed by documentation of a systematic increase in cAMP production by HEK293T‐LGR7 cells in response to increasing volumes of day 0 milk. Addition of lactation day 14 milk, porcine insulin, or human insulin‐like growth factor 1 to HEK293T‐LGR7 cells, or porcine RLX treatment of nontransfected HEK293T cells, failed to elicit a cAMP response. Western blot analysis of milk proteins revealed an 18‐kDa protein band, indicating that pro RLX is the primary form of bioactive RLX in porcine milk. Data support the lactocrine hypothesis and suggest a role for milk‐borne pro RLX in porcine neonatal development.

Nursing During the First Two Days of Life Is Essential for the Expression of Proteins Important for Growth and Remodeling of the Neonatal Porcine Cervix

The neonatal porcine cervix is sensitive to hormones, including relaxin (RLX), from birth. Whether nursing is required to establish the cervical developmental program or to determine cervical developmental trajectory is unknown. The objective of study 1 was to determine effects of age and nursing on expression of molecular markers and mediators of porcine cervical growth and remodeling from birth to postnatal day (PND) 2 and to document effects of RLX treatment during this period on expression of targeted gene products in nursed vs. replacer-fed gilts. Study 2 was conducted to determine effects of age at first nursing and duration of nursing from birth on expression of targeted transcripts or proteins at PND 14. Nursing supported cervical estrogen receptor-α, vascular endothelial growth factor, matrix metalloproteinase (MMP)9, and antiapoptotic B-cell lymphoma-2 protein expression on PND 2. These proteins were undetectable in replacer-fed gilts. Returning replacer-fed gilts to nursing after PND 2 did not restore cervical expression of these proteins by PND 14. RLX increased (P < 0.05) cervical estrogen receptor-α, vascular endothelial growth factor, and B-cell lymphoma-2 protein in nursed gilts, MMP2 protein in nursed and replacer-fed gilts, and decreased (P < 0.05) pro-MMP9 protein in nursed gilts, and RXFP1 mRNA levels in nursed and replacer-fed gilts at PND 2. Replacer feeding for 2 wk from birth increased (P < 0.05) RXFP1 mRNA levels on PND 14. Results support the lactocrine hypothesis for maternal programming of neonatal tissues. Nursing from birth is required to establish the neonatal cervical developmental program and to maintain cervical developmental trajectory to PND 14.

Mesangial cell apoptosis induced by stimulation of the adenosine A3 receptor: signaling and apoptotic events.

Mesangial cell apoptosis has been proposed as a means of resolution of glomerular hypercellularity in proliferative forms of glomerular disease. We previously demonstrated that adenosine causes mesangial cell apoptosis by stimulating the A3-type adenosine receptor. This is a G protein-coupled receptor shown to activate kinases involved in apoptotic signaling. In this work, we assessed changes in phosphorylation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2 and in levels of specific pro- and antiapoptotic proteins following exposure of mesangial cells to the A3 adenosine receptor agonist N(6)-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA). Cultured mesangial cells were incubated with IB-MECA for 30 minutes and 6, 24, and 48 hours. IB-MECA was used at a concentration (30 μM) that induces a reproducible degree of mesangial cell apoptosis. Changes in ERK1/2 phosphorylation and in protein levels of Bcl-2, Bax, and caspase 3 were assessed by Western blot analysis. IB-MECA markedly increased phosphorylation of ERK1/2. This effect peaked at 5 minutes, dissipated by 20 minutes, and was abolished by the inhibitor of ERK phosphorylation, compound U0126, in a dose-dependent manner. This inhibitor had no effect on the extent of IB-MECA-induced apoptosis. Bcl-2 levels progressively declined, whereas those of Bax and activated caspase 3 increased. These observations indicate that stimulation of the A3-type adenosine receptor causes mesangial cell apoptosis via mechanisms independent of ERK activation. The observations also point to an imbalance in the expression of antiapoptotic (Bcl-2) and proapoptotic (Bax, caspase 3) proteins as a potential mechanism underlying adenosine-induced mesangial cell apoptosis.

Maternal lactocrine programming of porcine reproductive tract development

The lactocrine hypothesis for maternal programming of female reproductive tract development is based on the idea that non‐nutritive, milk‐borne bioactive factors (MbFs), delivered from mother to offspring during nursing, play a role in determining the trajectory of development with long‐term consequences in the adult. Porcine female reproductive tract development is completed postnatally, and the period during which maternal support of neonatal growth derives exclusively from colostrum/milk defines a window of opportunity for lactocrine programming of reproductive tissues. Beyond nutrition, milk serves as a delivery system for a variety of bioactive factors. Porcine relaxin is a prototypical MbF. Present in colostrum at highest concentrations at birth, relaxin is transmitted into the circulation of nursing piglets where it can act on Relaxin receptors found in neonatal female reproductive tract tissues. This process is facilitated by the physiology of the maternal–neonatal dyad and the fact that the neonatal gastrointestinal tract is open to absorb macromolecules for a period of time postnatally. Age at first nursing and duration of nursing from birth are also important for porcine female reproductive tract development. These parameters affect both the quality and quantity of colostrum consumed. Disruption of lactocrine signaling by feeding milk replacer from birth altered porcine uterine, cervical, and testicular development by postnatal Day 2. Moreover, insufficient colostrum consumption in nursing piglets can impair uterine capacity to support viable litters of optimal size in adulthood. In the pig, lactocrine signaling supports neonatal organizational events associated with normal reproductive development and may program adult uterine capacity.