Neuropathic and inflammatory pain are modulated by tuberoinfundibular peptide of 39 residues.

Nociceptive information is modulated by a large number of endogenous signaling agents that change over the course of recovery from injury. This plasticity makes understanding regulatory mechanisms involved in descending inhibition of pain scientifically and clinically important. Neurons that synthesize the neuropeptide TIP39 project to many areas that modulate nociceptive information. These areas are enriched in its receptor, the parathyroid hormone 2 receptor (PTH2R). We previously found that TIP39 affects several acute nociceptive responses, leading us to now investigate its potential role in chronic pain. Following nerve injury, both PTH2R and TIP39 knockout mice developed less tactile and thermal hypersensitivity than controls and returned to baseline sensory thresholds faster. Effects of hindpaw inflammatory injury were similarly decreased in knockout mice. Blockade of α-2 adrenergic receptors increased the tactile and thermal sensitivity of apparently recovered knockout mice, returning it to levels of neuropathic controls. Mice with locus coeruleus (LC) area injection of lentivirus encoding a secreted PTH2R antagonist had a rapid, α-2 reversible, apparent recovery from neuropathic injury similar to the knockout mice. Ablation of LC area glutamatergic neurons led to local PTH2R-ir loss, and barley lectin was transferred from local glutamatergic neurons to GABA interneurons that surround the LC. These results suggest that TIP39 signaling modulates sensory thresholds via effects on glutamatergic transmission to brainstem GABAergic interneurons that innervate noradrenergic neurons. TIP39's normal role may be to inhibit release of hypoalgesic amounts of norepinephrine during chronic pain. The neuropeptide may help maintain central sensitization, which could serve to enhance guarding behavior.

Parathyroid hormone (PTH) and PTH-related peptide domains contributing to activation of different PTH receptor-mediated signaling pathways.

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), acting through the osteoblast PTH1 receptor (PTH1R), play important roles in bone remodeling. Intermittent administration of PTH(1-34) (teriparatide) leads to bone formation, whereas continuous administration paradoxically leads to bone resorption. Activation of PTH1R promotes regulation of multiple signaling pathways, including G(s)/cAMP/protein kinase A, G(q)/calcium/protein kinase C, ß-arrestin recruitment, and extracellular signal-related kinase (ERK)1/2 phosphorylation, as well as receptor internalization, but their role in promoting anabolic and catabolic actions of PTH(1-34) are unclear. In the present investigation, a collection of PTH(1-34) and PTHrP(1-34) peptide analogs were evaluated in orthogonal human PTH1R (hPTH1R) functional assays capturing G(s)- and G(q)-signaling, ß-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization to further define the patterns of PTH1R signaling that they stimulate and further establish peptide domains contributing to agonist activity. Results indicate that both N- and C-terminal domains of PTH and PTHrP are critical for activation of signaling pathways. However, modifications of both regions lead to more substantial decreases in agonist potency and efficacy to stimulate G(q)-signaling, ß-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization than to stimulate G(s)-signaling. The substantial contribution of the peptide C-terminal domain in activation of hPTH1R signaling suggests a role in positioning of the peptide N-terminal region into the receptor J-domain. Several PTH and PTHrP peptides evaluated in this study promote different patterns of biased agonist signaling and may serve as useful tools to further elucidate therapeutically relevant PTH1R signaling in osteoblasts. With a better understanding of therapeutically relevant signaling, novel biased peptides with desired signaling could be designed for safer and more effective treatment of osteoporosis.

TIP39/parathyroid hormone type 2 receptor signaling is a potent inhibitor of chondrocyte proliferation and differentiation.

Tuberoinfundibular peptide of 39 residues (TIP39) is a member of the parathyroid hormone (PTH) family of peptide hormones that exerts its function by interacting with the PTH type 2 receptor (PTH2R). Presently, no known function has been attributed to this signaling pathway in the developing skeleton. We observed that TIP39 and PTH2R were present in the newborn mouse growth plate, with the receptor localizing in the resting zone whereas ligand expression was restricted exclusively in prehypertrophic and hypertrophic chondrocytes. By 8 wk of life, PTH2R, and to a lesser degree TIP39, immunoreactivity was present in articular chondrocytes. We therefore sought to investigate the role of TIP39/PTH2R signaling in chondrocytes by generating stably transfected CFK2 chondrocytic cells overexpressing PTH2R (CFK2R). TIP39 treatment of CFK2R clones in culture inhibited their proliferation by restricting cells at the G(0)/G(1) phase of the cell cycle, coupled with decreased expression and activity of cyclin-dependent kinases Cdk2 and Cdk4, while p21, an inhibitor of Cdks, was upregulated. In addition, TIP39 treatment decreased expression of differentiation markers in these cells associated with marked alterations in extracellular matrix and metalloproteinase expression. Transcription of Sox9, the master regulator of cartilage differentiation, was reduced in TIP39-treated CFK2R clones. Moreover, Sox9 promoter activity, as measured by luciferase reporter assay, was markedly diminished after TIP39 treatment. In summary, our results show that TIP39/PTH2R signaling inhibits proliferation and alters differentiation of chondrocytes by modulating SOX9 expression, thereby substantiating the functional significance of this signaling pathway in chondrocyte biology.

Tuberoinfundibular peptide of 39 residues: a new mediator of cardiac function via nitric oxide production in the rat heart.

Tuberoinfundibular peptide (TIP39) was initially identified as a neurotransmitter and agonist of the PTH2 receptor, which is expressed in the cardiovascular system. This study documents for the first time the cardiac expression and function of TIP39. Expression was analyzed via RT-PCR. Function was characterized on Langendorff-perfused rat hearts as left ventricular developed pressure (LVDP) and on isolated cells via a cell edge detection system. cGMP levels were detected with RIA. Tuberoinfundibular peptide (TIP39) mRNA was found to be constitutively expressed in coronary endothelium cells, isolated cardiomyocytes, ventricles, atria, and aorta. At first we investigated the vasodilatory properties of TIP39 (100 nM) in the presence of L-nitro-arginine (L-NA, 100 microM). Surprisingly, TIP39 had no vasodilatory effect but decreased LVDP by 35 +/- 7%. In the absence of L-NA, addition of TIP39 decreased LVDP by 8 +/- 2%. The PTH2 receptor antagonist Trp23-Tyr36-PTHrP (1-36, 100 nM) abolished this TIP39 effect in the presence of L-NA. The experiments with isolated cardiomyocytes provided similar results. TIP39 (10 nM) lowered the contraction amplitude by 6 +/- 3%. In the presence of L-NA (100 micromol/liter), TIP39 lowered the amplitude by 34 +/- 6%. cGMP concentration in cardiomyocytes was stimulated by TIP39 (10 nM) in the same range as by the nitric oxide (NO) donor SNAP (100 microM). In the presence of L-NA, this increase was abolished. These results suggest that an inhibition of endogenous NO production unmasks a profound negative inotropic effect of TIP39 that is mediated by an activation of the PTH2 receptor. The results obtained with isolated cardiomyocytes suggest that myocyte-derived NO, rather than vascular NO, is responsible for this effect. cGMP seems to be the downstream signal of produced NO.

Novel G protein-coupled receptors as pain targets.

G protein-coupled receptors (GPCRs) and their ligands play a number of important roles in the modulation of acute and chronic pain. Indeed, opioid and cannabinoid ligands are of established therapeutic value for pain management, and further exploitation of the specific GPCR subtypes (delta-opioid, CB1 and CB2) for these ligands may yield more selective, potent analgesics with favorable side effects. More recent identification of a number of other GPCRs involved in pain pathways (eg, sensory neuron specific receptors) and selective ligands that modulate pain transmission, has highlighted further therapeutic opportunities. A further challenge to understanding pain modulation and an additional dimension for targeting analgesia is the discovery of GPCR heteromerization and accessory and regulatory proteins, such as regulator of G protein-signaling proteins, involved in expression and regulation of GPCR.

PTH2 receptor-mediated inhibitory effect of parathyroid hormone and TIP39 on cell proliferation.

The parathyroid hormone (PTH) has dual mitogenic and inhibitory effects on cell proliferation, depending on the cell type and experimental conditions. PTH can signal via two different receptors, both positively coupled to the adenylyl cyclase/cyclic AMP pathway which can mimic some of the proliferative effects of PTH. We evaluated the role of the type-2 PTH (PTH2) receptor on cell proliferation in clonal human embryonic kidney HEK293 cells stably expressing the human PTH2 receptor. Using a cyclic AMP-responsive gene-reporter, we confirmed that the tuberoinfundibular peptide (TIP39) and various human (h) PTH fragments including hPTH-(1-34) were potent agonists (EC(50) in the range of 0.01-0.3 nM) whereas the bovine (b) PTH peptides b(Tyr(34))PTH-(7-34) and its tryptophan derivative b[D-Trp(12),Tyr(34)]PTH-(7-34) behaved as antagonists (IC(50)=117 and 249 nM, respectively). hPTH-(1-34) produced a dose-dependent inhibition of cell proliferation (EC(50)=8.5+/-0.4 nM) after 3 days and this effect was fully reversed by the tryptophan derivative antagonist. The same effect was observed with TIP39 which caused a 30% maximal inhibition. These findings reveal that PTH2 receptor activation can inhibit cell proliferation and might explain the dual functionality of PTH on cell proliferation.

[Mechanism of intracerebral calcification in hypoparathyroidism].

The mechanism of intracranial calcification in hypoparathyroidism, more frequently seen in pseudo--than idiopathic hypoparathyroidism, has not been completely elucidated, but may be related more to the duration of hypocalcaemia and hyperphosphatemia than parathyroid hormone itself. Hyperphosphatemia promotes ectopic calcification, especially in blood vessel and periarticular tissue in renal failure, but in brain tissue in hypoparathyroidism. Participation of PTH receptor2 in the brain and superoxide production by mitochondria in hypoparathyroidism should be explored with reference to intracerebral calcification and neurodegenerative diseases.

Maternal absence of the parathyroid hormone 2 receptor affects postnatal pup development.

During the lactation period, mothers have a variety of adaptive changes in brain physiology and behaviour that allow them to properly raise their pups. The exact circuitries and mechanisms responsible for these changes are not fully understood. Recent evidence suggests that the neuropeptide tuberoinfundibular peptide of 39 residues (TIP39) and its receptor, the parathyroid hormone 2 receptor (PTH2-R), contribute to these mechanisms. To further investigate this idea, we evaluated the growth rate of pups from dams with a genetically inactivated PTH2-R (PTH2-R-KO), as well as maternal behavioural and neuroendocrine parameters. We observed that PTH2-R-KO-reared pups had a slowed growth rate. This was associated with a reduced volume of milk yielded by PTH2-R-KO dams after 30-min suckling compared to wild-type (WT) dams when pups were returned after 5 h of separation. Our data suggest a reduced sensitivity of PTH2-R-KO dams to pup stimulation. We also observed a significant reduction in suckling-induced c-Fos expression in the paraventricular hypothalamic nucleus and signs of lower prolactin levels in the PTH2-R-KO dams. Our data suggest that the reduced growth rate of PTH2-R-KO-reared pups was likely the result of alterations in the milk-production pathway rather than modifications in behaviour. Although PTH2-R-KO dams showed increased anxiety in the elevated zero-maze test, no differences from WT dams in maternal behaviour were observed. Taken together, our findings suggest the involvement of the TIP39/PTH2-R system in the pathways involved in the successful development of the pups.

The TIP39-PTH2 receptor system: unique peptidergic cell groups in the brainstem and their interactions with central regulatory mechanisms.

Tuberoinfundibular peptide of 39 residues (TIP39) is the recently purified endogenous ligand of the previously orphan G-protein coupled parathyroid hormone 2 receptor (PTH2R). The TIP39-PTH2R system is a unique neuropeptide-receptor system whose localization and functions in the central nervous system are different from any other neuropeptides. TIP39 is expressed in two brain regions, the subparafascicular area in the posterior thalamus, and the medial paralemniscal nucleus in the lateral pons. Subparafascicular TIP39 neurons seem to divide into a medial and a lateral cell population in the periventricular gray of the thalamus, and in the posterior intralaminar complex of the thalamus, respectively. Periventricular thalamic TIP39 neurons project mostly to limbic brain regions, the posterior intralaminar thalamic TIP39 neurons to neuroendocrine brain areas, and the medial paralemniscal TIP39 neurons to auditory and other brainstem regions, and the spinal cord. The widely distributed axon terminals of TIP39 neurons have a similar distribution as the PTH2R-containing neurons, and their fibers, providing the anatomical basis of a neuromodulatory action of TIP39. Initial functional studies implicated the TIP39-PTH2R system in nociceptive information processing in the spinal cord, in the regulation of different hypophysiotropic neurons in the hypothalamus, and in the modulation of affective behaviors. Recently developed novel experimental tools including mice with targeted mutations of the TIP39-PTH2R system and specific antagonists of the PTH2R will further facilitate the identification of the specific roles of TIP39 and the PTH2R.