Prolactin Family Research Articles

Searching for the genetic basis of impaired placentation in the Dahl salt sensitive rat model of superimposed preeclampsia

Preeclampsia, a hypertensive disorder of pregnancy, is a leading cause of maternal and fetal morbidity and mortality worldwide. Despite the rising prevalence and severity of this disease, the initiating factors responsible for the development of preeclampsia remain unknown. While it is hypothesized that impaired placentation precedes the maternal syndrome of preeclampsia, specific factors that contribute to altered vascular remodeling at the maternal‐fetal interface have not yet been identified. We have recently characterized the Dahl salt sensitive rat (Dahl S) on a 0.3% NaCl diet as a spontaneous model of superimposed preeclampsia, and here we use this model to investigate potential genetic factors that contribute to the impaired placental vasculogenesis we have observed in this model. Sprague Dawley (SD) rats were used for comparison as a model of healthy pregnancy and normal placentation. Female Dahl S and SD rat strains were each mated, and implantation sites in the uterus were isolated on gestational days (GD) 7, 10, and 14 (n=4–5/group). Uterine samples from virgin (GD0) rats were also obtained, and placental samples were isolated on GD14 (n=5/group). RNA was isolated, whole transcriptome analysis was performed using Affymetrix GeneChips, and data analyzed using GeneSifter and Ingenuity Pathway Analysis (IPA) software. A preliminary analysis of microarray data (fold‐change >1.5; p<0.05) between SD and Dahl S showed a number of genes differentially expressed at GD 0 (466 genes), 7 (714), 10 (679), and 14 (512). At GD 0–10, few genes [e.g., fibronectin type III domain containing 7 and Cd86 (involved in T‐cell activation and proliferation)] were significantly different using more stringent B&H FDR (P<0.05), suggesting there are small/relatively subtle changes between the strains that lead to strong physiological difference. However, by GD14 (the beginning of the “third trimester” in the rat), 33 genes were significantly different between SD and S using more stringent statistical criteria. This analysis revealed greater expression of genes involved in cellular responses to hypoxia and oxidative stress (e.g. lactoperoxidase and prolactin family 8, subfamily a, member 2) in the Dahl S rat. Using a 2‐way ANOVA to look at changes that occur between groups and over time (strain X time interaction), gene differences between strains were 466 (FDR, p<0.05); with time were 1727 (FDR; p<0.001); and for strain X time there were only two [mast cell protease 10 and zinc metallopeptidase, STE24 homolog (S. cerevisiae) with most stringent FDR]. When the statistical criteria was relaxed (p<0.05), the number of strain X time genes was much greater (n=847). Currently, we are working to perform a more systematic analysis, including identification of major biological functions, canonical pathways, and gene networks to place the gene differences in context of physiological changes. The identification of these specific pathways that lead to impaired placentation in this model of preeclampsia will provide clues to understanding the pathogenesis of the disease.

A Prolactin Family Paralog Regulates Placental Adaptations to a Physiological Stressor.

The prolactin (PRL) family of hormones and cytokines participates in the regulation of optimal reproductive performance in the mouse and rat. Members of the PRL family are expressed in the anterior pituitary, uterus, and/or placenta. In the present study, we investigated the ontogeny of PRL family 7, subfamily b, member 1 (PRL7B1; also called PRL-like protein-N, PLP-N) expression in the developing mouse placenta and established a mouse model for investigating the biological function of PRL7B1. Transcripts for Prl7b1 were first detected on Gestation Day (d) 8.5. From gestation d8.5 through d14.5, Prl7b1 was expressed in trophoblast cells residing at the interface between maternal mesometrial decidua and the developing placenta. On gestation d17.5, the predominant cellular source of Prl7b1 mRNA was migratory trophoblast cells invading into the uterine mesometrial decidua. The Prl7b1 null mutant allele was generated via replacement of the endogenous Prl7b1 coding sequence with beta-galactosidase (LacZ) reporter and neomycin cassettes. The mutant Prl7b1 allele was successfully passed through the germline. Homozygous Prl7b1 mutant mice were viable and fertile. Under standard animal housing conditions, Prl7b1 had undetectable effects on placentation and pregnancy. Hypoxia exposure during pregnancy evoked adaptations in the organization of the wild-type placenta that were not observed in Prl7b1 null placentation sites. In summary, PRL7B1 is viewed as a part of a pathway regulating placental adaptations to physiological stressors.

Formation of fetal opioid system

The article presented literature review about of endogenous opioid system (EOS) formation consist of opioid receptors complex and its ligands (endogenous opioid peptides) in different tissues including placenta. It was shown that formation of fetal EOS is going with anatomic and functional development of the central nervous system and EOS expression begins in the placental tissues as soon as implantation and starts till the end of the pregnancy. Influence of opioid peptides on secretion progesterone, prolactin family peptides, growth hormone, placental lactogens and prolypherine from the trophoblast tissue is discussed.

Overexpression of PRL7D1 in Leydig Cells Causes Male Reproductive Dysfunction in Mice.

Prolactin family 7, subfamily d, member 1 (PRL7D1) is found in mouse placenta. Our recent work showed that PRL7D1 is also present in mouse testis Leydig cells, and the expression of PRL7D1 in the testis exhibits an age-related increase. In the present study, we generated transgenic mice with Leydig cell-specific PRL7D1 overexpression to explore its function during male reproduction. Prl7d1 male mice exhibited subfertility as reflected by reduced sperm counts and litter sizes. The testes from Prl7d1 transgenic mice appeared histologically normal, but the frequency of apoptotic germ cells was increased. Prl7d1 transgenic mice also had lower testosterone concentrations than wild-type mice. Mechanistic studies revealed that Prl7d1 transgenic mice have defects in the testicular expression of steroidogenic acute regulatory protein (STAR) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase cluster (HSD3B). Further studies revealed that PRL7D1 overexpression affected the expression of transferrin (TF) in Sertoli cells. These results suggest that PRL7D1 overexpression could lead to increased germ cell apoptosis and exert an inhibitory effect on testosterone production in Leydig cells by reducing the expression of certain steroidogenic-related genes. In addition, PRL7D1 appears to have important roles in the function of Sertoli cells, which, in turn, affects male fertility. We conclude that the expression level of PRL7D1 is associated with the reproductive function of male mice.

Production and Purification of Recombinant Somatolactin and its Effects on Insulin-like Growth Factors Gene Expression in Tilapia Hepatocytes

Somatolactin (SL), the member of the growth hormone (GH)/prolactin (PRL) family, is fish-specific pituitary hormone with diverse functions. However, little is known about its biological function in fish hepatocytes. Using tilapia as a model, SLtranscripts were shown to be widely expressed in various extrapituitary tissues with the relatively high expression level in liver. To explore the biological action of SL in hepatocytes, we produced and purified recombinant tilapia SL protein which could induce pigment aggregation in tilapia melanophores. Further, the antiserum for the SL was produced and its specificity was confirmed by antiserum preabsorption. During 4 week starvation, hepatic SL transcripts in starved fish were significantly higher than the control fish starting on the 1st week of starvation until 4th week. After re-feeding, the SL transcripts level returned to normal. Using primary cultures of tilapia hepatocytes, insulin-like growth factors (IGF1 and IGF2) gene expression were elevated by static incubation with recombinant tilapia SL. In contrast, removal endogenous SL by immunoneutralization using SL antiserum was shown to inhibit IGF1 and IGFgene expression. These findings, taken together, provide evidence for the first time that SL may serve as a novel regulator in fish stimulating IGF1 and IGF gene expression in hepatocytes.

Photoactivation of ROS Production In Situ Transiently Activates Cell Proliferation in Mouse Skin and in the Hair Follicle Stem Cell Niche Promoting Hair Growth and Wound Healing

The role of reactive oxygen species (ROS) in the regulation of hair follicle (HF) cycle and skin homeostasis is poorly characterized. ROS have been traditionally linked to human disease and aging, but recent findings suggest that they can also have beneficial physiological functions in vivo in mammals. To test this hypothesis, we transiently switched on in situ ROS production in mouse skin. This process activated cell proliferation in the tissue and, interestingly, in the bulge region of the HF, a major reservoir of epidermal stem cells, promoting hair growth, as well as stimulating tissue repair after severe burn injury. We further show that these effects were associated with a transient Src kinase phosphorylation at Tyr416 and with a strong transcriptional activation of the prolactin family 2 subfamily c of growth factors. Our results point to potentially relevant modes of skin homeostasis regulation and demonstrate that a local and transient ROS production can regulate stem cell and tissue function in the whole organism.

Identification of target genes for a prolactin family paralog in mouse decidua.

Prolactin family 8, subfamily a, member 2 (PRL8A2; also called decidual prolactin-related protein; dPRP) is a member of the expanded prolactin family. PRL8A2 is expressed in the uterine decidua and contributes to pregnancy-dependent adaptations to hypoxia. The purpose of this study was to identify gene targets for PRL8A2 action within the uteroplacental compartment. Affymetrix DNA microarray analysis was performed for RNA samples from WT and Prl8a2 null tissues. Validation of the DNA microarray was performed using quantitative RT-PCR. Nine genes were confirmed with decreased expression in Prl8a2 null tissues (e.g., Klk7, Rimklb, Arhgef6, Calm4, Sprr2h, Prl4a1, Ccl27, Lipg, and Htra3). These include potential decidual, endothelial and trophoblast cell targets positively regulated by PRL8A2. A significant upregulation of Derl3, Herpud1, Creld2, Hsp90b1, Ddit3 and Hspa5 was identified in Prl8a2 null tissues, reflecting an increased endoplasmic reticulum (ER) stress response. ER stress genes were prominently expressed in the uterine decidua. We propose that PRL8A2 is a mediator of progesterone-dependent modulation of intrauterine responses to physiological stressors.

A positive feedback loop between prolactin and STAT5 promotes angiogenesis.

The signal transduction events that orchestrate cellular activities required for angiogenesis remain incompletely understood. We and others recently described that proangiogenic mediators such as fibroblast growth factors can activate members of the signal transducers and activators of transcription (STAT) family. STAT5 activation is necessary and sufficient to induce migration, invasion and tube formation of endothelial cells. STAT5 effects on endothelial cells require the secretion of the prolactin (PRL) family member proliferin-1 (PLF1) in mice and PRL in humans. In human endothelial cells, PRL activates the PRL receptor (PRLR) resulting in MAPK and STAT5 activation, thus closing a positive feedback loop. In vivo, endothelial cell-derived PRL is expected to combine with PRL of tumor cell and pituitary origin to raise the concentration of this polypeptide hormone in the tumor microenvironment. Thus, PRL may stimulate tumor angiogenesis via autocrine, paracrine, and endocrine pathways. The disruption of tumor angiogenesis by interfering with PRL signaling may offer an attractive target for therapeutic intervention.

Kisspeptin induction of somatolactin-α release in goldfish pituitary cells: functional role of cAMP/PKA-, PLC/PKC-, and Ca(2+)/calmodulin-dependent cascades.

Although the importance of kisspeptin in the pituitary is firmly established, the signaling mechanisms for the pituitary actions of kisspeptin are still largely unknown. Somatolactin (SL), a member of the growth hormone (GH)/prolactin (PRL) family, is a pituitary hormone with pleiotropic functions in fish, but its regulation by kisspeptin has not been examined. To investigate the functional role of kisspeptin in SL regulation, expression of two paralogues of goldfish Kiss1 receptors (Kiss1ra and Kiss1rb) were confirmed in immunoidentified SLα but not SLβ cells isolated by RT-PCR coupled with laser capture microdissection. In goldfish pituitary cells prepared from neurointermediate lobe (NIL), synthetic goldfish Kiss decapeptides (gKiss1-10 and gKiss2-10) could increase SLα release. Consistent with the lack of Kiss1r expression in SLβ cells, SLβ release was not altered by kisspeptin stimulation. In parallel experiments, goldfish gKiss1-10 could elevate cyclic adenosine monophosphate (cAMP) production, upregulate protein kinase A (PKA) and protein kinase C (PKC) activities, and trigger a rapid rise in intracellular Ca(2+) levels in goldfish NIL cells. Using a pharmacological approach, cAMP/PKA and phospholipase C (PLC)/PKC pathways and subsequent activation of Ca(2+)/calmodulin (CaM)-dependent cascades were shown to be involved in SLα release induced by gKiss1-10. Apparently, the Ca(2+)-dependent cascades were triggered by extracellular Ca(2+) entry via voltage-sensitive Ca(2+) channels and mobilization of inositol trisphosphate-sensitive intracellular Ca(2+) stores. Our results demonstrate that gKiss1-10 can act directly at the pituitary level to trigger SLα release via a complex network of post-receptor signaling mechanisms.

Evidence of placental endoplasmic reticulum stress in the pathophysiology of gestational diabetes mellitus

Evidence of placental endoplasmic reticulum stress in the pathophysiology of gestational diabetes mellitus

Placental prolactin family levels are modified during gestation in the diabetic rat

Placental prolactin family levels are modified during gestation in the diabetic rat

Placental antiangiogenic prolactin fragments are increased in human and rat maternal diabetes

Introduction/objectivesThe role of the placenta in diabetic mothers on fetal development and programming is unknown. Prolactin (PRL) produced by decidual endometrial cells may have an impact. Although full-length PRL is angiogenic, the processed form by bone morphogenetic protein-1 (BMP-1) and/or cathepsin D (CTSD) is antiangiogenic.The objectives were to investigate the involvement of decidual PRL and its antiangiogenic fragments in placentas from type-1 diabetic women (T1D) and from pregnant diabetic rats with lower offspring weights than controls. MethodsPRL, BMP-1, and CTSD gene expressions and PRL protein level were assessed in T1D placentas (n=8) at delivery and compared to controls (n=5). Wistar rats received, at day 7 of pregnancy, streptozotocin (STZ) (n=5) or nicotinamide (NCT) plus STZ (n=9) or vehicle (n=9). Placental whole-genome gene expression and PRL western blots were performed at birth. ResultsIn human placentas, PRL (p<0.05) and BMP-1 (p<0.01) gene expressions were increased with a higher amount of cleaved PRL (p<0.05) in T1D than controls. In rats, diabetes was more pronounced in STZ than in NCT–STZ group with intra-uterine growth restriction. Decidual prolactin-related protein (Dprp) (p<0.01) and Bmp-1 (p<0.001) genes were up-regulated in both diabetic groups, with an increased cleaved PRL amount in the STZ (p<0.05) and NCT–STZ (p<0.05) groups compared to controls. No difference in CTSD gene expression was observed in rats or women. ConclusionsAlterations in the levels of the PRL family are associated with maternal diabetes in both rats and T1D women suggesting that placental changes in these hormones impact on fetal development.

Localization and expression patterns of prolactin-like protein J in mouse testis.

Prolactin (PRL)‑like proteinJ (PLP‑J) is a member of the prolactin family, mainly expressed in the placental decidua tissues of females, and is involved in gestation. To the best of our knowledge, it has not previously been shown to be expressed in males. Preliminary experiments of the present study indicated that PLP‑J is expressed in the testis of male mice and is implicated in the regulation of testicular function. To definitively address whether PLP‑J is expressed in the mouse testis, the expression pattern and cellular localization of PLP‑J in mouse testes during postnatal development were characterized in the current study using molecular and immunological methods. Reverse transcription (RT)‑polymerase chain reaction (PCR) was performed to amplify gene fragments from mouse testis specimens, which yielded sequences matching those of the PLP‑J gene in Genbank. Subsequently, insituhybridization showed that PLP‑J was localized in interstitial tissue of the mouse testis. Immunofluorescence results indicated that PLP‑J and 3β‑hydroxysteroid dehydrogenase1 were colocalized in testis Leydig cells, confirming PLP‑J expression in Leydig cells. In addition, PLP‑J gene expression levels were examined at different stages of postnatal mouse development in male testis tissues using quantitative RT‑PCR and western blotting. The results revealed that PLP‑J expression levels were lowest in 18‑day‑old mice and highest in adults aged 4months. Levels observed in 16‑month‑old individuals were lower than those observed in the 4‑month‑old mice, but remained significantly higher than the levels observed in 18‑day‑old mice. Furthermore, the roles of PLP‑J in the murine testis TM3Leydig cell line were studied. The results demonstrated that the upregulation of PLP‑J expression in TM3Leydig cells did not affect testosterone production or the cell cycle. In conclusion, this study demonstrated that PLP‑J, a known member of the PRL family that was previously considered to be expressed solely in females, is also expressed in the testis of males with an age‑dependent expression profile. Nevertheless, the physiological role of PLP‑J in males remains unclear.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates release of somatolactin (SL)-α and SL-β from cultured goldfish pituitary cells via the PAC1 receptor-signaling pathway, and affects the expression of SL-α and SL-β mRNAs

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that stimulates the release of adenohypophyseal hormone from the pituitary in fish. In the goldfish, PACAP induces the release of somatolactin (SL), in particular, from cultured pituitary cells. SL belongs to the growth hormone and prolactin family, and comprises two molecular variants termed SL-α and SL-β in goldfish. However, there is no information about the involvement of PACAP in the regulation of SL-α and SL-β release and the expression of their mRNAs. Therefore, we examined the effect of PACAP on SL-α and SL-β release from cultured goldfish pituitary cells. Treatment with PACAP (10−10–10−7M) increased the release of both SL-α and SL-β. The stimulatory action of PACAP (10−9M) on SL-α and SL-β release was blocked by treatment with a PACAP-selective receptor (PAC1R) antagonist, PACAP(6–38) (10−6M). We also examined whether PACAP affects the expression of SL-α and SL-β mRNAs in cultured pituitary cells. Treatment with PACAP (10−9 and 10−8M) for 6h decreased the expression level of SL-α mRNA but increased that of SL-β mRNA. The action of PACAP (10−8M) on SL-β mRNA expression was blocked by treatment with PACAP(6–38) (10−6M), whereas PACAP(6–38) elicited no change in the expression of SL-α mRNA. These results indicate that in cultured goldfish pituitary cells, PACAP stimulates the release of SL-α and SL-β, and expression of SL-β mRNA, via the PAC1R-signaling pathway. However, the mechanism whereby PACAP inhibits the expression of SL-α mRNA does not seem to be mediated by PAC1R signaling.

Biology of the Placental Proteins in Domestic Ruminants: Expression, Proposed Roles and Practical Applications

The placenta, a lodgment at the feto-maternal interface, is a temporal organ that plays crucial roles in maintaining gestation and fetal development. To achieve these purposes, the placenta produces an array of proteins with distinct spatio-temporal characteristics. In this review, the authors focus on the paralog families of prolactin (PRL) and aspartic proteinase, exclusively produced by the placenta, and discuss their biological roles and practical significance in animal husbandry. The bovine placental PRL family consists of one classical member and at least ten non-classical members. Bovine placental lactogen (PL) is a unique classical member due to its lactogenic activity and potentially involved in partitioning nutrients to maintain fetal development. In contrast, the biological roles of non-classical members in bovine placental PRL family proteins remain unclear. Recent papers have reported that a non-classical member protein named prolactin-related protein -I (PRP-I) exhibited angiogenic activity following C-terminal cleavage by proteolytic enzymes. These results suggest that non-classical members of the placental PRL family exert their biological activities via specific mechanisms other than the PRL receptor-signaling pathway. The bovine genome contains a hundred or more aspartic proteinase-like genes and at least 22 mRNAs with close structural relationships are transcribed in the placenta. These molecules correspond to pregnancy-specific protein (PSP)-B, PSP-60 and pregnancy-associated glycoprotein (PAG) as reported previously. The true character of PSPs and PAG is thought to be a mixture of placental aspartic proteinase-like proteins. Immunological detection of PAG protein in maternal serum is the basis for early pregnancy diagnosis in ruminants. Currently, early pregnancy diagnosis by PAG assay is available for cows, buffaloes and deer.

反芻家畜の胎盤血管の新生調節機構に関わるタンパク質：胎盤性プロラクチンファミリータンパク質に注目して

反芻家畜の胎盤血管の新生調節機構に関わるタンパク質：胎盤性プロラクチンファミリータンパク質に注目して

Signal transduction mechanisms for autocrine/paracrine regulation of somatolactin-α secretion and synthesis in carp pituitary cells by somatolactin-α and -β

Pituitary hormones can act locally via autocrine/paracrine mechanisms to modulate pituitary functions, which represents an interesting aspect of pituitary regulation other than the traditional hypothalamic input and feedback signals from the periphery. Somatolactin, a member of the growth hormone (GH)/prolactin (PL) family, is a pleiotropic hormone with diverse functions, but its pituitary actions are still unknown. Recently, two SL isoforms, SLα and SLβ, have been cloned in grass carp. Based on the sequences obtained, recombinant proteins of carp SLα and SLβ with similar bioactivity in inducing pigment aggregation in carp melanophores were produced. In carp pituitary cells, SLα secretion and cell content were elevated by static incubation with recombinant carp SLα and SLβ, respectively. These stimulatory actions occurred with a parallel rise in SLα mRNA level with no changes in SLβ secretion, cell content, and gene expression. In contrast, SLα mRNA expression could be reduced by removing endogenous SLα and SLβ with immunoneutralization. At the pituitary cell level, SLα release, cell content, and mRNA expression induced by carp SLα and SLβ could be blocked by inhibiting JAK2/STAT5, PI3K/Akt, MEK1/2, and p38 MAPK, respectively. Furthermore, SLα and SLβ induction also triggered rapid phosphorylation of STAT5, Akt, MEK1/2, ERK1/2, MKK3/6, and p38 MAPK. These results suggest that 1) SLα and SLβ produced locally in the carp pituitary can serve as novel autocrine/paracrine stimulators for SLα secretion and synthesis and 2) SLα production induced by local release of SLα and SLβ probably are mediated by the JAK2/STAT5, PI3K/Akt, and MAPK signaling pathways.

Abstract 203: Transcriptomic Analysis of Placental Chorionic Villi During Placental Ischemia-induced Hypertension

While the precise etiology of preeclampsia is unclear, it is believed that a central causative agent of the disorder is placental ischemia caused by placental hypoperfusion and chronic ischemia. This results in the secretion of pathogenic factors into the maternal circulation. While a number of these factors have been identified (i.e. sFlt-1, AT1-AA, inflammatory cytokines), the full pathogenic profile remains unresolved. Here we have attempted to identify novel biomarkers and pathogenic factors in the ischemic placenta by quantitative microarray analysis of both mRNA and miRNA expression in a rodent model of chronic placental ischemia, the Reduced Uterine Perfusion Pressure (RUPP) model. Gene expression arrays from chorionic villi from control and RUPP animals (n=6 each) revealed 2557 differentially expressed genes at a statistical cutoff of p&lt;0.05 (1140 up-regulated, 1417 down-regulated. The thirty most differentially affected transcripts had fold changes of 2.6-1.4 and included several genes previously identified in the placenta of human patients and experimental models, including pappalysin-2, CEACAM1, and heme oxygenase-1. Most pronounced was a significant increase in the expression of 9 prolactin family members, indicating a massive upregulation of the prolactin system. In addition, a number of novel pro-inflammatory and anti-angiogenic targets were identified. The expression of miRNA in these samples was also determined, and at a statistical cutoff of p&lt;0.05 there were 577 down-regulated and 458 up-regulated miRNAs, of which 73 were changed by more than 50%. Among the pathways known to be affected by these miRNA’s are the VEGF signaling pathways, HMOX1, the Notch pathway, and the HIF activator p300, all of which have previously been implicated in the etiology of preeclampsia. Functional analysis of these transcripts could provide important mechanistic insight into the regulatory mechanisms behind the development of preeclampsia. Together, these data provide interesting biomarkers for placental ischemia and could suggest new therapeutic targets for the treatment of preeclampsia.

Angiogenesis Induced by Signal Transducer and Activator of Transcription 5A (STAT5A) Is Dependent on Autocrine Activity of Proliferin

Multiple secreted factors induce the formation of new blood vessels (angiogenesis). The signal transduction events that orchestrate the numerous cellular activities required for angiogenesis remain incompletely understood. We have shown previously that STAT5 plays a pivotal role in angiogenesis induced by FGF2 and FGF8b. To delineate the signaling pathway downstream of STAT5, we expressed constitutively active (CA) or dominant-negative (DN) mutant STAT5A in mouse brain endothelial cells (EC). We found that the conditioned medium from CA-STAT5A but not from dominant-negative STAT5A overexpressing EC is sufficient to induce EC invasion and tube formation, indicating that STAT5A regulates the secretion of autocrine proangiogenic factors. Conversely, CA-STAT5A-induced conditioned medium had no effect on EC proliferation. Using a comparative genome-wide transcription array screen, we identified the prolactin family member proliferin (PLF1 and PLF4) as a candidate autocrine factor. The CA-STAT5A-dependent transcription and secretion of PLF by EC was confirmed by quantitative RT-PCR and Western blotting, respectively. CA-STAT5A binds to the PLF1 promoter region, suggesting a direct transcriptional regulation. Knockdown of PLF expression by shRNA or by blocking of PLF activity with neutralizing antibodies removed the CA-STAT5A-dependent proangiogenic activity from the conditioned medium of EC. Similarly, the ability of concentrated conditioned medium from CA-STAT5A transfected EC to induce angiogenesis in Matrigel plugs in vivo was abolished when PLF was depleted from the medium. These observations demonstrate a FGF/STAT5/PLF signaling cascade in EC and implicate PLF as autocrine regulator of EC invasion and tube formation.

CATALYTIC RECEPTORS

CATALYTIC RECEPTORS