Emerging roles of atypical chemokine receptor 3 (ACKR3) in normal development and physiology.

The discovery that atypical chemokine receptors (ACKRs) can initiate alternative signaling pathways rather than classical G-protein coupled receptor (GPCR) signaling has changed the paradigm of chemokine receptors and their roles in modulating chemotactic responses. The ACKR family has grown over the years, with discovery of new functions and roles in a variety of pathophysiological conditions. However, the extent to which these receptors regulate normal physiology is still continuously expanding. In particular, atypical chemokine receptor 3 (ACKR3) has proven to be an important receptor in mediating normal biological functions, including cardiac development and migration of cortical neurons. In this review, we illustrate the versatile and intriguing role of ACKR3 in physiology.

Cardiovascular effects of exogenous adrenomedullin and CGRP in Ramp and Calcrl deficient mice.

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are potent vasodilator peptides and serve as ligands for the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CLR/Calcrl). Three GPCR accessory proteins called receptor activity-modifying proteins (RAMPs) modify the ligand binding affinity of the receptor such that the CLR/RAMP1 heterodimer preferably binds CGRP, while CLR/RAMP2 and CLR/RAMP3 have a stronger affinity for AM. Here we determine the contribution of each of the three RAMPs to blood pressure control in response to exogenous AM and CGRP by measuring the blood pressure of mice with genetic reduction or deletion of the receptor components. Thus, the cardiovascular response of Ramp1-/-, Ramp2+/-, Ramp3-/-, Ramp1-/-/Ramp3-/- double-knockout (dKO), and Calcrl+/- mice to AM and CGRP were compared to wildtype mice. While under anesthesia, Ramp1-/- male mice had significantly higher basal blood pressure than wildtype males; a difference which was not present in female mice. Additionally, anesthetized Ramp1-/-, Ramp3-/-, and Calcrl+/- male mice exhibited significantly higher basal blood pressure than females of the same genotype. The hypotensive response to intravenously injected AM was greatly attenuated in Ramp1-/- mice, and to a lesser extent in Ramp3-/- and Calcrl+/- mice. However, Ramp1-/-/Ramp3-/- dKO mice retained some hypotensive response to AM. These results suggest that the hypotensive effect of AM is primarily mediated through the CLR/RAMP1 heterodimer, but that AM signaling via CLR/RAMP2 and CLR/RAMP3 also contributes to some hypotensive action. On the other hand, CGRP's hypotensive activity seems to be predominantly through the CLR/RAMP1 heterodimer. With this knowledge, therapeutic AM or CGRP peptides could be designed to cause less hypotension while maintaining canonical receptor-RAMP mediated signaling.

Midregional pro-adrenomedullin plasma concentrations are blunted in severe preeclampsia.

Levels of the peptide hormone adrenomedullin (AM) are elevated during normal pregnancy, but whether this differs during complications of pregnancy remains unresolved. AM can be quantified by measuring its pre-prohormone byproduct, midregional pro-adrenomedullin (MR-proADM). MR-proADM has shown prognostic value as a biomarker of heart failure, sepsis, and community-acquired pneumonia. Given the relevance of AM to pregnancy, we tested the hypothesis that MR-proADM provides a biomarker for preeclampsia. We find that MR-proADM plasma concentrations are blunted in severe preeclampsia and that MR-proADM is similarly effective as established biomarkers endoglin and placental growth factor at discriminating patients with severe preeclampsia from controls.

Receptor activity modifying proteins (RAMPs) interact with the VPAC2 receptor and CRF1 receptors and modulate their function.

BACKGROUND AND PURPOSE: Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-activating peptide 2 receptor (VPAC(2)) and the type 1 corticotrophin releasing factor receptor (CRF(1)) has been examined. EXPERIMENTAL APPROACH: GPCRs were co-transfected with RAMPs in HEK 293S and CHO-K1 cells. Cell surface expression of RAMPs and GPCRs was examined by ELISA. Where there was evidence for interactions, agonist-stimulated cAMP production, Ca(2+) mobilization and GTPγS binding to G(s), G(i), G(12) and G(q) were examined. The ability of CRF to stimulate adrenal corticotrophic hormone release in Ramp2(+/-) mice was assessed. KEY RESULTS: The GLP-1 receptor failed to enhance the cell surface expression of any RAMP. VPAC(2) enhanced the cell surface expression of all three RAMPs. CRF(1) enhanced the cell surface expression of RAMP2; the cell surface expression of CRF(1) was also increased. There was no effect on agonist-stimulated cAMP production. However, there was enhanced G-protein coupling in a receptor and agonist-dependent manner. The CRF(1) : RAMP2 complex resulted in enhanced elevation of intracellular calcium to CRF and urocortin 1 but not sauvagine. In Ramp2(+/-) mice, there was a loss of responsiveness to CRF. CONCLUSIONS AND IMPLICATIONS: The VPAC(2) and CRF(1) receptors interact with RAMPs. This modulates G-protein coupling in an agonist-specific manner. For CRF(1), coupling to RAMP2 may be of physiological significance.

Toll-like receptor 4 mediates intrauterine growth restriction after systemic Campylobacter rectus infection in mice.

Campylobacter rectus is associated with fetal exposure and low birthweight in humans. C. rectus also invades placental tissues and induces fetal intrauterine growth restriction (IUGR) in mice, along with overexpression of Toll-like receptors (TLR4), suggesting that TLR4 may mediate placental immunity and IUGR in mice. To test this hypothesis we examined the effect of in vitro TLR4 neutralization on trophoblastic proinflammatory activity and studied the IUGR phenotype in a congenic TLR4-mutant mouse strain after in vivo C. rectus infection. Human trophoblasts were pretreated with TLR4 neutralizing antibodies and infected with C. rectus; proinflammatory cytokine production was assessed by cytokine multiplex assays. Neutralizing TLR4 antibodies significantly impaired the production of proinflammatory cytokines in trophoblastic cells after infection in a dose-dependent manner. We used a subcutaneous chamber model to provide a C. rectus challenge in BALB/cAnPt (TLR4(Lps-d) ) and wild-type (WT) females. Females were mated with WT or TLR4(Lps-d) males once/week; pregnant mice were infected at (E)7.5 and sacrificed at (E)16.5 to establish IUGR phenotypes. Maternal C. rectus infection significantly decreased fetal weight/length in infected WT when compared with sham WT controls (P < 0.05, analysis of variance). However, infected TLR4(Lps-d -/-) mice did not show statistically significant differences in fetal weight and length when compared with WT controls (P > 0.05). Furthermore, heterozygous TLR4(Lps-d +/-) fetuses showed IUGR phenotype rescue. We conclude that TLR4 is an important mediator of trophoblastic proinflammatory responses and TLR4-deficient fetuses do not develop IUGR phenotypes after C. rectus infection, suggesting that placental cytokine activation is likely to be mediated by TLR4 during low birthweight/preterm birth pathogenesis.

Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional Adrenomedullin gene.

Adrenomedullin, a recently identified potent vasodilator, is expressed widely and has been suggested to have functions ranging from reproduction to blood pressure regulation. To elucidate these functions and define more precisely sites of Adm expression, we replaced the coding region of the Adm gene in mice with a sequence encoding enhanced green fluorescent protein while leaving the Adm promoter intact. We find that Adm(-/-) embryos die at midgestation with extreme hydrops fetalis and cardiovascular abnormalities, including overdeveloped ventricular trabeculae and underdeveloped arterial walls. These data suggest that genetically determined absence of Adm may be one cause of nonimmune hydrops fetalis in humans.

Developmental roles of the steroidogenic acute regulatory protein (StAR) as revealed by StAR knockout mice.

Steroidogenic acute regulatory protein (StAR) is essential for adrenal and gonadal steroidogenesis, stimulating the translocation of cholesterol to the inner mitochondrial membrane where steroidogenesis commences. StAR mutations in humans cause congenital lipoid adrenal hyperplasia (lipoid CAH), an autosomal recessive condition with severe deficiencies of all classes of steroid hormones. We previously described StAR knockout mice that mimic many features of lipoid CAH patients. By keeping StAR knockout mice alive with corticosteroid replacement, we now examine the temporal effects of StAR deficiency on the structure and function of steroidogenic tissues. The adrenal glands, affected most severely at birth, exhibited progressive increases in lipid deposits with aging. The testes of newborn StAR knockout mice contained scattered lipid deposits in the interstitial region, presumably in remnants of fetal Leydig cells. By 8 weeks of age, the interstitial lipid deposits worsened considerably and were associated with Leydig cell hyperplasia. Despite these changes, germ cells in the seminiferous tubules appeared intact histologically, suggesting that the StAR knockout mice retained some capacity for androgen biosynthesis. Sperm maturation was delayed, and the germ cells exhibited histological features of apoptosis, consistent with suboptimal androgen production. Immediately after birth, the ovaries of StAR knockout mice appeared normal. After the time of normal puberty, however, prominent lipid deposits accumulated in the interstitial region, accompanied by marked luteinization of stromal cells and incomplete follicular maturation that ultimately culminated in premature ovarian failure. These studies provide the first systematic evaluation of the developmental consequences of StAR deficiency in the various steroidogenic organs.

Targeted disruption of StAR provides novel insights into congenital adrenal hyperplasia.

To explore the function of StAR in a system that can be experimentally manipulated, and to develop a mouse model for the human disorder lipoid congenital adrenal hyperplasia (lipoid CAH), we used targeted gene disruption to produce a mouse line deficient in StAR protein. Initially, StAR knockout mice were indistinguishable from wildtype littermates, except that all had female external genitalia. After birth, they showed signs of either respiratory distress or volume depletion and eventually died. Hormone assays confirmed severe defects in adrenal steroids, whereas hormones constituting the gonadal axis did not differ significantly from levels in wildtype littermates. Histologically, the adrenal cortex of StAR knockout mice contained florid lipid deposits, as visualized with oil red O stain. Lesser lipid deposits were observed in the steroidogenic compartment of the testis and none in the ovary. The sex-specific differences in gonadal involvement provide evidence for a two-stage model of the pathogenesis of StAR deficiency, with trophic hormone stimulation causing progressive accumulation of lipids within the steroidogenic cells which ultimately kills them. These StAR knockout mice provide a novel system in which to study StAR's essential roles in adrenocortical and gonadal steroidogenesis.

Targeted disruption of the mouse gene encoding steroidogenic acute regulatory protein provides insights into congenital lipoid adrenal hyperplasia.

An essential component of regulated steroidogenesis is the translocation of cholesterol from the cytoplasm to the inner mitochondrial membrane where the cholesterol side-chain cleavage enzyme carries out the first committed step in steroidogenesis. Recent studies showed that a 30-kDa mitochondrial phosphoprotein, designated steroidogenic acute regulatory protein (StAR), is essential for this translocation. To allow us to explore the roles of StAR in a system amenable to experimental manipulation and to develop an animal model for the human disorder lipoid congenital adrenal hyperplasia (lipoid CAH), we used targeted gene disruption to produce StAR knockout mice. These StAR knockout mice were indistinguishable initially from wild-type littermates, except that males and females had female external genitalia. After birth, they failed to grow normally and died from adrenocortical insufficiency. Hormone assays confirmed severe defects in adrenal steroids-with loss of negative feedback regulation at hypothalamic-pituitary levels-whereas hormones constituting the gonadal axis did not differ significantly from levels in wild-type littermates. Histologically, the adrenal cortex of StAR knockout mice contained florid lipid deposits, with lesser deposits in the steroidogenic compartment of the testis and none in the ovary. The sex-specific differences in gonadal involvement support a two-stage model of the pathogenesis of StAR deficiency, with trophic hormone stimulation inducing progressive accumulation of lipids within the steroidogenic cells and ultimately causing their death. These StAR knockout mice provide a useful model system in which to determine the mechanisms of StAR's essential roles in adrenocortical and gonadal steroidogenesis.

Characterization of the promoter region of the mouse gene encoding the steroidogenic acute regulatory protein.

Steroidogenic acute regulatory protein (StAR) delivers cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme carries out the first committed step in steroid hormone biosynthesis. StAR expression is restricted to steroidogenic cells and is rapidly induced by treatment with trophic hormones or cAMP. We analyzed the 5'-flanking region of the mouse StAR gene to elucidate the mechanisms that regulate its cell-specific and hormone-induced expression. In transient transfection assays, a luciferase reporter gene driven by the StAR 5'-flanking region was preferentially expressed by steroidogenic Y1 adrenocortical and MA-10 Leydig cells in a cAMP-responsive manner. 5'-Deletion and site-directed mutagenesis studies identified a region between -254 and -113 that is essential for full levels of promoter activity. This region contains a binding site for the orphan nuclear receptor steroidogenic factor-1 (SF-1) that, although not required for hormone induction, is critical for basal promoter activity, thus implicating SF-1 in StAR expression. Analyses of knockout mice deficient in SF-1 further supported an important role for SF-1 in StAR gene expression. These studies provide novel insights into the mechanisms that regulate StAR gene expression and extend our understanding of SF-1's global roles within steroidogenic cells.

Steroidogenic factor 1 plays multiple roles in endocrine development and function.

The nuclear hormone receptor family comprises a group of structurally related transcriptional regulators that mediate the actions of diverse ligands, including steroid hormones, thyroid hormone, vitamin D, and retinoids. The nuclear receptor family also contains members for which activating ligands have not been identified-the orphan nuclear receptors. One of these orphan nuclear receptors, steroidogenic factor 1 (SF-1), has emerged as an essential regulator of steroidogenic cell function within the adrenal cortex and gonads; SF-1 also plays important roles in reproduction at all three levels of the hypothalamic-pituitary-gonadal axis. First identified as a tissue-specific regulator of the transcription of the cytochrome P450 steroid hydroxylases, considerably broader roles for SF-1 were revealed by genetic studies in mice lacking SF-1 due to targeted gene disruption. These SF-1-knockout mice had agenesis of their adrenal glands and gonads, male-to-female sex reversal of their internal and external genitalia, impaired gonadotrope function, and agenesis of the ventromedial hypothalamic nucleus. These studies delineated essential roles of SF-1 in regulating endocrine differentiation and function at multiple levels. Despite these insights into roles of SF-1, the precise mechanisms by which SF-1 exerts its multiple effects remain to be determined. This review highlights experiments that have established SF-1 as a pivotal determinant of endocrine differentiation and function and identifies areas in which additional studies are needed to expand our understanding of SF-1 action.

Steroidogenic factor 1 acts at all levels of the reproductive axis.

The conversion of cholesterol into steroid hormones occurs through the sequential actions of the cytochrome P450 steroid hydroxylases. Attempts to understand the mechanisms responsible for the temporal and spatial expression patterns of these enzymes led to the identification of a shared regulator, termed steroidogenic factor 1 (SF-1). SF-1 coordinately regulates the steroid hydroxylase genes and thus functions as a global mediator of steroidogenesis. Of greater significance, recent studies using a knockout mouse model have further implicated SF-1 in a variety of processes ranging from development of the steroidogenic organs to the normal function of gonadotropes and the development of the ventromedial hypothalamic nucleus. A fundamental aspect of elucidating the role of SF-1 at all levels of the reproductive axis is to identify its cell-specific target genes. The recent purification and cloning of the steroidogenic acute regulatory (StAR) protein has provided an intriguing new candidate through which SF-1 acts to mediate its effects on reproductive competence. These studies yield novel insights into the processes of steroidogenesis, endocrine development, and reproductive function.

Hormonal and developmental regulation of the steroidogenic acute regulatory protein.

A crucial event in the acute regulation of steroidogenesis by trophic hormones is the delivery of cholesterol into the mitochondria where it is converted to pregnenolone by the cholesterol side chain cleavage enzyme. Although considerable controversy exists regarding the exact mechanisms that underlie this acute response to hormone stimulation, recent studies suggest that the Steroidogenic Acute Regulatory (StAR) protein, a hormone-induced 30-kilodalton mitochondrial protein, plays an essential role. We now extend these studies by establishing in MA-10 mouse Leydig tumor cells a temporal relationship between levels of StAR expression and steroidogenesis in response to hormone stimulation. These data indicate that trophic hormones regulate StAR mRNA and protein within a time frame concomitant with the acute production of steroid hormones and provide the first evidence implicating changes in StAR transcription and/or mRNA stability in the functional response of steroidogenic cells to hormone action. In addition, in situ hybridization analyses of StAR expression in embryonic and adult mice demonstrated a precise spatial and temporal relationship in vivo between StAR expression and the capacity to produce steroid hormones. These experiments strengthen considerably the evidence that StAR is the key mediator of the acute induction of steroidogenesis and provide new insights into the mechanisms by which trophic hormones activate steroidogenesis in steroidogenic cells.