XerD-mediated FtsK-independent integration of TLC? into the Vibrio cholerae genome

As in most bacteria, topological problems arising from the circularity of the two Vibrio cholerae chromosomes, chrI and chrII, are resolved by the addition of a crossover at a specific site of each chromosome, dif, by two tyrosine recombinases, XerC and XerD. The reaction is under the control of a cell division protein, FtsK, which activates the formation of a Holliday Junction (HJ) intermediate by XerD catalysis that is resolved into product by XerC catalysis. Many plasmids and phages exploit Xer recombination for dimer resolution and for integration, respectively. In all cases so far described, they rely on an alternative recombination pathway in which XerC catalyzes the formation of a HJ independently of FtsK. This is notably the case for CTXϕ, the cholera toxin phage. Here, we show that in contrast, integration of TLCϕ, a toxin-linked cryptic satellite phage that is almost always found integrated at the chrI dif site before CTXϕ, depends on the formation of a HJ by XerD catalysis, which is then resolved by XerC catalysis. The reaction nevertheless escapes the normal cellular control exerted by FtsK on XerD. In addition, we show that the same reaction promotes the excision of TLCϕ, along with any CTXϕ copy present between dif and its left attachment site, providing a plausible mechanism for how chrI CTXϕ copies can be eliminated, as occurred in the second wave of the current cholera pandemic.The causative agent of the epidemic severe diarrheal disease cholera is the Vibrio cholerae bacterium. A major determinant of its pathogenicity, the cholera enterotoxin, is encoded in the genome of the filamentous cholera toxin phage, CTXϕ (1). Like many other V. cholerae filamentous phages, CTXϕ uses a host chromosomally encoded, site-specific recombination (Xer) machinery for lysogenic conversion (2–4). The Xer machinery normally serves to resolve chromosome dimers, which result from homologous recombination events between the two chromatids of circular chromosomes during or after replication. In V. cholerae, as in most bacteria, the Xer machinery consists of two tyrosine recombinases, XerC and XerD. They act at a unique specific chromosomal site, dif, on each of the two circular chromosomes, chrI and chrII, of the bacterium (5). Integrative mobile elements exploiting Xer (IMEXs) carry a dif-like site on their circular genome, attP (3, 4) (Fig. 1A). XerC and XerD promote their integration by catalyzing a recombination event between this site and a cognate chromosomal dif site (3, 4) (Fig. 1A). Based on the structure of their attP site, IMEXs can be grouped into at least three families (3, 4) (Fig. 1B). In all cases, however, a new functional dif site is restored after integration, which permits multiple successive integration events (Fig. 1A). Indeed, most clinical and environmental V. cholerae isolates harbor large IMEX arrays (6, 7).Open in a separate windowFig. 1.Systems that use Xer. (A) Scheme depicting the sequential integration of IMEXs. Triangles represent attP and dif sites, pointing from the XerD binding site to the XerC binding site. Chromosomal DNA (black), TLCϕ DNA (blue), and CTXϕ DNA (magenta) are indicated. Dotted triangles represent nonfunctional CTXϕ sites. (B) Sequence alignment of dif1, attP^CTX, attP^VGJ, attP^TLC, difA, and dif2. Bases differing from dif1 are indicated in color. Bases that do not fit the XerD binding site consensus are indicated in lowercase. XerC (●) and XerD (○) cleavage points are indicated. (C) Xer recombination pathways. XerC (light gray circles), XerD (dark gray circles), dif sites (red and black lines), and attP^CTX and attP^VGJ (magenta and green lines) are indicated. XerC and XerD catalysis-suitable conformations are depicted as horizontal and vertical synapses, respectively. Cleavage points are indicated as in B.IMEX array formation participates in the continuous and rapid dissemination of new cholera toxin variants in at least three ways. First, CTXϕ integration is intrinsically irreversible because the active form of its attP site consists of the stem of a hairpin of its ssDNA genome, which is masked in the host dsDNA genome (8, 9) (Fig. 1 A and B). However, free CTXϕ genome copies can be produced by a process analogous to rolling circle replication after the integration of a second IMEX harboring the same integration/replication machinery, such as the RS1 satellite phage, which permits the production of new CTXϕ viral particles (10). Second, the V. cholerae Gillermo Javier filamentous phage (VGJϕ) belongs to a second category of IMEXs whose attP site permits cycles of integration and excision by Xer recombination (11). VGJϕ excision allows for the formation of hybrid molecules harboring the concatenated genomes of CTXϕ and VGJϕ, provided that VGJϕ integrated before CTXϕ (11). The hybrid molecules can be packaged into VGJϕ particles. VGJϕ particles have a different receptor than CTXϕ, which permits transduction of the cholera toxin genes to cells that do not express the receptor of CTXϕ (11–13). Finally, integration of the toxin-linked cryptic phage (TLCϕ), a satellite phage that defines a third category of IMEXs, seems to be a prerequisite to the toxigenic conversion of many V. cholerae strains (14, 15). IMEXs from this family are found integrated in the genome of many bacteria outside of the Vibrios, including human, animal, and plant pathogens, which sparked considerable interest in the understanding of how they exploit the Xer machinery at the molecular level (3, 4).Xer recombination sites consist of 11-bp XerC and XerD binding arms, separated by an overlap region at the border of which recombination occurs (Fig. 1B). XerC and XerD each promote the exchange of a specific pair of strands (Fig. 1B). Recombination between dif sites is under the control of a cell division protein, FtsK, which restricts it temporally to the time of constriction and spatially to a specific zone within the terminus region of chromosomes (16–19). FtsK triggers the formation of a Holliday junction (HJ) by XerD catalysis, which is converted into product by XerC catalysis after isomerization (20, 21) (Fig. 1C). The intermediate HJ is stable enough to be converted into product by replication when XerC catalysis is impeded (5, 17) (Fig. 1C). The integration of IMEXs of the CTXϕ and VGJϕ families escapes FtsK control. The lack of homology in the overlap regions of their attP sites and the dif sites they target prevents any potential XerD-mediated strand exchange (Fig. 1B). CTXϕ and VGJϕ rely on the exchange of a single pair of strands by XerC catalysis for integration, with the resulting HJ being converted into product by replication (8, 9, 11) (Fig. 1C). In the case of CTXϕ, integration is facilitated by an additional host factor, EndoIII, which impedes futile cycles of XerC catalysis once the pseudo-HJ is formed (22) (Fig. 1C). In contrast, the overlap region of TLCϕ attP, attP^TLC, is fully homologous to the overlaps of dif1 and difA, the two sites in which it was found to be integrated (Fig. 1B). Four integration pathways could thus be considered, depending on whether recombination is initiated by XerC or XerD catalysis, and whether it ends with a second pair of strand exchange or not. In addition, attP^TLC lacks a consensus XerD binding site, which could affect the whole recombination process (Fig. 1B).Here, we show that attP^TLC is a poor XerD binding substrate. Nevertheless, we show that TLCϕ integration is initiated by XerD catalysis and that the resulting HJ is converted into product by XerC catalysis. We further show that TLCϕ integration is independent of FtsK. Finally, we demonstrate that the same reaction can lead to the excision of TLCϕ–CTXϕ arrays, providing a plausible mechanism for how all of the CTXϕ copies integrated on V. cholerae chrI can be eliminated in a single step, as occurred in ancestors of strains from the second wave of the current cholera pandemic (23–25).     

Structural insight into the molecular mechanism of allosteric activation of human cystathionine β-synthase by S-adenosylmethionine

Cystathionine β-synthase (CBS) is a heme-dependent and pyridoxal-5′-phosphate–dependent protein that controls the flux of sulfur from methionine to cysteine, a precursor of glutathione, taurine, and H₂S. Deficiency of CBS activity causes homocystinuria, the most frequent disorder of sulfur amino acid metabolism. In contrast to CBSs from lower organisms, human CBS (hCBS) is allosterically activated by S-adenosylmethionine (AdoMet), which binds to the regulatory domain and triggers a conformational change that allows the protein to progress from the basal toward the activated state. The structural basis of the underlying molecular mechanism has remained elusive so far. Here, we present the structure of hCBS with bound AdoMet, revealing the activated conformation of the human enzyme. Binding of AdoMet triggers a conformational change in the Bateman module of the regulatory domain that favors its association with a Bateman module of the complementary subunit to form an antiparallel CBS module. Such an arrangement is very similar to that found in the constitutively activated insect CBS. In the presence of AdoMet, the autoinhibition exerted by the regulatory region is eliminated, allowing for improved access of substrates to the catalytic pocket. Based on the availability of both the basal and the activated structures, we discuss the mechanism of hCBS activation by AdoMet and the properties of the AdoMet binding site, as well as the responsiveness of the enzyme to its allosteric regulator. The structure described herein paves the way for the rational design of compounds modulating hCBS activity and thus transsulfuration, redox status, and H₂S biogenesis.Cystathionine β-synthase (CBS; EC 4.2.1.22) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of the hydroxyl group of l-serine (Ser) by l-homocysteine (Hcy), yielding cystathionine (Cth) (1). A deficient activity of human CBS (hCBS) is the cause of classical homocystinuria [CBS-deficient homocystinuria (CBSDH); Online Mendelian Inheritance in Man (OMIM) no. 236200], an autosomal, recessive inborn error of sulfur amino acid metabolism, characterized by increased levels of Hcy in plasma and urine. CBSDH manifests as a combination of connective tissue defects, skeletal deformities, vascular thrombosis, and mental retardation (2).The hCBS is a homotetrameric enzyme whose subunits are organized into three structural domains. The N-terminal region binds heme and is thought to function in redox sensing and/or enzyme folding (3, 4). The central catalytic core shows the fold of the type II family PLP-dependent enzymes (5, 6). Finally, the C-terminal region consists of a tandem pair of CBS motifs (7–9) that bind S-adenosylmethionine (AdoMet) and lead to an increase in catalytic activity by up to fivefold (10, 11). The CBS motif pair, commonly known as a “Bateman module” (12, 13), is responsible for CBS subunit tetramerization (14, 15). The presence of pathogenic missense mutations in this region often does not impair enzyme activity but typically interferes with binding of AdoMet and/or the enzyme’s activation by AdoMet (15–17). Removal of the regulatory region leads to a dimer with much increased activity (14, 15). Recently, we showed that removal of residues 516–525, forming a flexible loop of the CBS2 motif of hCBS, yields dimeric species (hCBSΔ516–525) with intact AdoMet binding capacity and activity responsiveness to AdoMet similar to a native hCBS WT (18).hCBS is regulated by a complex molecular mechanism that remains poorly understood. More than a decade ago, we and others hypothesized that hCBS might exist in two different conformations: a “basal” state with low activity, where the C-terminal regulatory domain would restrict the access of substrates into the catalytic site, and an AdoMet-bound “activated” state, where the AdoMet-induced conformational change would allow for enzyme activation (16, 19). Recently, we have unveiled the relative orientations of the regulatory and catalytic domains in hCBS (18), which were in a striking contrast to those of both the previous in silico models (20, 21) and the Drosophila melanogaster (dCBS) structure (22). Our data showed that, although the pairing mode and the orientation of catalytic cores are similar in both insect dCBS and hCBS, the position of their regulatory domains is markedly different (18). In the basal state, the Bateman modules from each hCBS unit are far apart and do not interact with each other, being placed just above the entrance of the catalytic site of the complementary subunit, thus hampering the access of substrates into this cavity. Our hCBSΔ516–525 structure additionally revealed the presence of two major cavities in the Bateman module, S1 and S2, one of which (S2) is solvent-exposed and probably represents the primary binding site for AdoMet (18). These findings are in agreement with the much higher basal activity of dCBS and its inability to bind or to be regulated by AdoMet (23, 24) and suggest that the structural basis underlying the regulation of the human enzyme markedly differs from CBS regulation in insects or yeast (24). Taken together, the available data indicate that binding of AdoMet to the Bateman module weakens the interaction between the regulatory domain and the catalytic core although the mechanism and the magnitude of the underlying structural effect are still under debate (16, 19, 25–27).To solve the molecular mechanism of hCBS regulation by AdoMet, we have analyzed the crystals of an engineered hCBSΔ516–525 protein that bears the mutation E201S, which potentially weakens and/or disrupts the interaction between the Bateman module and the catalytic core (Fig. 1A), thus favoring the activation of the enzyme. The data presented here fill a long-sought structural gap by unraveling the crystal structure of AdoMet-bound hCBS, thus providing the overall fold of the enzyme in its activated conformation and the identity of the AdoMet binding sites. Comparison with the structures of hCBS in basal conformation and constitutively activated dCBS was instrumental in the understanding of the regulatory role played by the C-terminal domain as well as the effect of some of the pathogenic mutations in the activation and/or inhibition of this key molecule of transsulfuration.Open in a separate windowFig. 1.Interactions between protein domains in basal hCBS. (A) In hCBSΔ516–525, residues Y484, N463, and S466 anchor the Bateman module (blue) to the protein core (gray) through H-bonds with the residues E201 and D198 from the loop L191–202, thus occluding the entrance to the catalytic pocket. (B) The CBS-specific activity of selected hCBS variants in the absence (blue bars) and the presence (red bars) of 300 µM AdoMet. hCBS enzyme species marked with “Δ” lack residues 516–525 and form dimers.     

Inhibition of the β-barrel assembly machine by a peptide that binds BamD

The protein complex that assembles integral membrane β-barrel proteins in the outer membranes of Gram-negative bacteria is an attractive target in the development of new antibiotics. This complex, the β-barrel assembly machine (Bam), contains two essential proteins, BamA and BamD. We have identified a peptide that inhibits the assembly of β-barrel proteins in vitro by characterizing the interaction of BamD with an unfolded substrate protein. This peptide is a fragment of the substrate protein and contains a conserved amino acid sequence. We have demonstrated that mutations of this sequence in the full-length substrate protein impair the protein’s assembly, implying that BamD’s interaction with this sequence is an important part of the assembly mechanism. Finally, we have found that in vivo expression of a peptide containing this sequence causes growth defects and sensitizes Escherichia coli to antibiotics to which they are normally resistant. Therefore, inhibiting the binding of substrates to BamD is a viable strategy for developing new antibiotics directed against Gram-negative bacteria.Membrane proteins with β-barrel structure are found in the outer membranes (OMs) of Gram-negative bacteria and in the mitochondria and chloroplasts of eukaryotes. These proteins are assembled into their native membranes by conserved protein complexes, which contain a β-barrel protein of the outer membrane protein 85 (Omp85) family and some number of accessory proteins (1–6). β-barrel assembly is an essential process and, as such, is an attractive target for the development of new antibiotics that could kill Gram-negative pathogens. However, the mechanism by which β-barrel assembly proceeds is only beginning to be elucidated. Structures of the components of the bacterial β-barrel assembly machine (BamA–E) have been determined recently, and several hypotheses about how they facilitate the assembly of its substrates have been proposed (7–17). The β-barrel component BamA contains a kinked β-strand, which might allow substrate proteins to be inserted by passing laterally from the lumen of the BamA β-barrel into the hydrophobic membrane (18). BamA also contains a large soluble domain in addition to its integral β-barrel; this region extends into the periplasmic space between the inner membrane and OM and consists of five polypeptide transport-associated (POTRA) domains (19). The POTRA domains bind the other four components of the assembly complex, BamB–E, which are lipoproteins. Structural and biochemical studies have suggested that the POTRA domains, BamB, and BamD may interact with substrate proteins during their assembly (13, 15, 19–22). However, more detailed information is required about the nature of those interactions and whether they are critical in the assembly process to develop an integrated mechanistic model.Only two components of the Bam complex, BamA and BamD, are essential for Escherichia coli viability (2, 23). BamD is found in all proteobacteria (24, 25), but it bears no obvious homology to any of the accessory components of the mitochondrial or chloroplastic complexes. It is therefore unclear what its essential function is and whether that function is specific to bacteria. Do the β-barrel assembly complexes in all species use the same mechanism (perhaps with different accessory proteins), or have they evolved different essential components to meet their organism-specific requirements? We have previously demonstrated that BamD binds to two unfolded OMP substrates, OmpA and BamA, and that it directly facilitates the assembly of BamA (21). In this study, we have characterized this interaction to understand its role in the assembly process carried out by the complete Bam complex and to assess whether interfering with the binding of substrates to BamD might be a viable strategy for developing new antibiotics.     

First Report of a Dominantly Inherited β-Thalassemia Caused by a Novel Elongated β-Globin Chain

A distinct set of mutations on the β-globin gene leads to dominantly inherited β-thalassemia (β-thal) that is associated with a disease phenotype in a single mutant copy. We described molecular and hematological characteristics of a novel elongated β-globin chain in combination with a known hemoglobin (Hb) variant (N-Baltimore or HBB: c.286A>G) in cis. The highly unstable Hb variant caused typical features of β-thal major (β-TM) or β-thal intermedia (β-TI) in two members of a family depending on their α-globin genotypes. The β mutant allele of the mother was transmitted in an autosomal dominant fashion to her daughter. They resemble severe forms of β-thal due to ineffective erythropoiesis. Taken together with previously published data, this result indicates that a dominant form of β-thal should be regarded as a phenotypic term of hemoglobinopathies caused by β chain variants that are highly unstable.     

“Diversion” Colitis Caused by Clostridium difficile Infection: Report of a Case

Recurrent ulcerative colitis and/or diversion colitis occur commonly in the rectal remnant after colectomy for ulcerative colitis. We report a case in which such a patient's symptoms of rectal discharge were initially thought to be the result of one or both of these diagnoses, on both clinical and histologic grounds. However, examination of the discharge revealed Clostridium difficile infection. Treatment with metronidazole suppositories improved his symptoms and avoided further inappropriate intervention. Effie Tsironi, M.D. is supported by the Hellenic Society of Gastroenterology, Greece. Reprints are not available.     

NF-κB inhibits osteogenic differentiation of mesenchymal stem cells by promoting β-catenin degradation

Mesenchymal stem cell (MSC)-based transplantation is a promising therapeutic approach for bone regeneration and repair. In the realm of therapeutic bone regeneration, the defect or injured tissues are frequently inflamed with an abnormal expression of inflammatory mediators. Growing evidence suggests that proinflammatory cytokines inhibit osteogenic differentiation and bone formation. Thus, for successful MSC-mediated repair, it is important to overcome the inflammation-mediated inhibition of tissue regeneration. In this study, using genetic and chemical approaches, we found that proinflammatory cytokines TNF and IL-17 stimulated IκB kinase (IKK)–NF-κB and impaired osteogenic differentiation of MSCs. In contrast, the inhibition of IKK–NF-κB significantly enhanced MSC-mediated bone formation. Mechanistically, we found that IKK–NF-κB activation promoted β-catenin ubiquitination and degradation through induction of Smurf1 and Smurf2. To translate our basic findings to potential clinic applications, we showed that the IKK small molecule inhibitor, IKKVI, enhanced osteogenic differentiation of MSCs. More importantly, the delivery of IKKVI promoted MSC-mediated craniofacial bone regeneration and repair in vivo. Considering the well established role of NF-κB in inflammation and infection, our results suggest that targeting IKK–NF-κB may have dual benefits in enhancing bone regeneration and repair and inhibiting inflammation, and this concept may also have applicability in many other tissue regeneration situations.     

Enhanced degradation of proteins of the basal lamina and stroma by matrix metalloproteinases from the salivary glands of Sjögren's syndrome patients: correlation with reduced structural integrity of acini and ducts

OBJECTIVE: To determine the effect of matrix metalloproteinase (MMP) activity from the labial salivary glands (LSGs) of Sj?gren's syndrome (SS) patients on proteins of the extracellular matrix (ECM) that form the basal lamina and stroma, and to compare this effect with the structural integrity of acini and ducts as well as the functionality of the LSGs. METHODS: Gelatinase activity was determined by zymography. The digestion pattern of extracellular matrix (ECM) macromolecules was detected by gel electrophoresis and quantified by densitometry. The structural integrity of acini and ducts was evaluated by light and electron microscopy. Secretory function was evaluated by measuring unstimulated salivary flow and by scintigraphy. RESULTS: LSG extracts showed increased levels of proteolytic activity toward purified proteins of the basal lamina (laminin and type IV collagen) and stroma (types I and III collagen and fibronectin). Enhanced degradation was most evident for fibronectin, laminin, and type IV collagen. Analysis of the ultrastructure of the acinar and ductal basal lamina revealed abnormalities ranging from disorganization to disappearance of this ECM structure. These changes were paralleled by an important loss of microvilli on the apical surface, as well as decreased unstimulated salivary flow. Interestingly, the results were similar in LSGs from all SS patients, regardless of the proximity of infiltrating mononuclear cell foci. CONCLUSION: Our observation that the proteolytic action of MMPs toward ECM macromolecules is increased in SS patients provides a rationale for understanding the dramatic changes in the structural organization observed in the basal lamina and apical surface of acini in these patients. The results provide new evidence that acinar and ductal cells from the LSGs of SS patients display a molecular potential, with increased capacity to markedly disorganize their ECM environment and, thus, damage their architecture and functionality.     

Intrahepatic Enhanced Expression of β2-Microglobulin Conformational Epitope in Acute Liver Allograft Rejection Evidence of Modulation by Glucocorticoids

The mechanisms by which glucocorticoids areeffective in acute liver rejection therapy are notentirely clear. The aims of this study were tocharacterize the intrahepatic immunological phenotype inacute liver rejection, as well as the effect ofglucocorticoids on cytokinestimulated hepatocyte celllines. Biopsy sections from these patients were studiedby immunohistochemistry. Cytokine-stimulated hepatocyte cell lines treated with glucocorticoids wereevaluated by flow cytometry. The intrahepatic expressionof both ₂-microglobulin conformationalepitope and intercellular adhesion molecule-1 was higher in acute rejection than in resolving rejection.Interestingly, glucocorticoids were able to modulate invitro the cytokineinduced expression of these moleculeson hepatocyte cell lines. Beneficial effects of the glucocorticoid treatment appear to beassociated with a modulation of a₂-microglobulin conformational epitopeand the intercellular adhesion molecule-1 onintrahepatic cellular targets in the acute rejection process.     

Lethal human neutropenic entercolitis caused by Clostridium chauvoei in the United States: tip of the iceberg?

The patient is a 44-year-old woman with metastatic grade 3 intra-ductal carcinoma of the breast who was started on palliative chemotherapy (docetaxel) 10 days prior to admission and presented to the emergency center complaining of diffuse abdominal pain and generalized weakness. CT abdomen showed diffuse bowel wall thickening from the cecum to the transverse colon with free fluid in the pelvis. The patient was neutropenic on admission (absolute neutrophil count of 600 cells/μl). She received antibiotics for 21 days for neutropenic enterocolitis. Blood culture isolate from admission was sent for 16s rRNA gene sequencing, which identified Clostridium chauvoei. While C. chauvoei has a long history of veterinary importance, this is the first documented case of infection caused by C. chauvoei in a human in the United States. C. chauvoei has a close phylogenetic relationship with C. septicum making the two species difficult to differentiate using conventional microbiologic methods. With increased use of more reliable detection methods the actual prevalence of C. chauvoei causing human disease may be higher than currently recognized.     

Thalassaemia intermedia caused by coinheritance of a β-thalassaemia mutation and a de novo duplication of α-globin genes in the paternal allele

Next generation sequencing identified a de novo, 204 kb, tandem duplication (αααα²⁰⁴) in the α-globin gene cluster of a Chinese thalassaemia intermedia patient. Haplotype analysis showed that the duplicated chromosome was of paternal origin. Molecular analysis of genomic DNA from the patient's lymphocytes, hair follicles, buccal mucosa cells, his father's lymphocytes and sperm cells excluded the possibility of somatic or germinal mosaicism. The analysis also indicated that this duplication arose during spermatogenesis. The microhomology in the breakpoint was found and suggested that this duplication could be formed by a coupled homologous and non-homologous recombination mechanism.     

Insight into the mechanism of an iron dioxygenase by resolution of steps following the FeIV═O species

Iron oxygenases generate elusive transient oxygen species to catalyze substrate oxygenation in a wide range of metabolic processes. Here we resolve the reaction sequence and structures of such intermediates for the archetypal non-heme Fe^II and α-ketoglutarate-dependent dioxygenase TauD. Time-resolved Raman spectra of the initial species with ¹⁶O¹⁸O oxygen unequivocally establish the Fe^IV═O structure. ¹H/²H substitution reveals direct interaction between the oxo group and the C1 proton of substrate taurine. Two new transient species were resolved following Fe^IV═O; one is assigned to the ν_FeO mode of an Fe^III─O(H) species, and a second is likely to arise from the vibration of a metal-coordinated oxygenated product, such as Fe^II─O─C₁ or Fe^II─OOCR. These results provide direct insight into the mechanism of substrate oxygenation and suggest an alternative to the hydroxyl radical rebinding paradigm.     

Anabolic action of parathyroid hormone regulated by the β2-adrenergic receptor

Parathyroid hormone (PTH), the major calcium-regulating hormone, and norepinephrine (NE), the principal neurotransmitter of sympathetic nerves, regulate bone remodeling by activating distinct cell-surface G protein-coupled receptors in osteoblasts: the parathyroid hormone type 1 receptor (PTHR) and the β(2)-adrenergic receptor (β(2)AR), respectively. These receptors activate a common cAMP/PKA signal transduction pathway mediated through the stimulatory heterotrimeric G protein. Activation of β(2)AR via the sympathetic nervous system decreases bone formation and increases bone resorption. Conversely, daily injection of PTH (1-34), a regimen known as intermittent (i)PTH treatment, increases bone mass through the stimulation of trabecular and cortical bone formation and decreases fracture incidences in severe cases of osteoporosis. Here, we show that iPTH has no osteoanabolic activity in mice lacking the β(2)AR. β(2)AR deficiency suppressed both iPTH-induced increase in bone formation and resorption. We showed that the lack of β(2)AR blocks expression of iPTH-target genes involved in bone formation and resorption that are regulated by the cAMP/PKA pathway. These data implicate an unexpected functional interaction between PTHR and β(2)AR, two G protein-coupled receptors from distinct families, which control bone formation and PTH anabolism.