Serine protease HTRA1 antagonizes transforming growth factor-ß signaling by cleaving its receptors and loss of HTRA1 in vivo enhances bone formation.

HTRA1 is a member of the High Temperature Requirement (HTRA1) family of serine proteases, which play a role in several biological and pathological processes. In part, HTRA1 regulation occurs by inhibiting the TGF-ß signaling pathway, however the mechanism of inhibition has not been fully defined. Previous studies have shown that HTRA1 is expressed in a variety of tissues, including sites of skeletal development. HTRA1 has also been implicated in the process of bone formation, although the precise manner of regulation is still unknown. This study investigated how HTRA1 regulates TGF-ß signaling and examined the in vivo effects of the loss of HTRA1. We demonstrated that recombinant HTRA1 was capable of cleaving both type II and type III TGF-ß receptors (TßRII and TßRIII) in vitro in a dose-dependent manner, but it did not affect the integrity of TßRI or TGF-ß. Overexpression of HTRA1 led to decreased levels of both TßRII and III on the cell surface but had no effect on TßRI. Silencing HTRA1 expression significantly increased TGF-ß binding to the cell surface and TGF-ß responsiveness within the cell. To examine the role of HTRA1 in vivo, we generated mice with a targeted gene deletion of HTRA1. Embryonic fibroblasts isolated from these mice displayed an increase in TGF-ß-induced expression of several genes known to promote bone formation. Importantly, the loss of HTRA1 in the knockout mice resulted in a marked increase in trabecular bone mass. This study has identified a novel regulatory mechanism by which HTRA1 antagonizes TGF-ß signaling, and has shown that HTRA1 plays a key role in the regulation of bone formation.

Disposition of human recombinant lubricin in naive rats and in a rat model of post-traumatic arthritis after intra-articular or intravenous administration.

We have recently demonstrated that intra-articular (IA) administration of human recombinant lubricin, LUB:1, significantly inhibited cartilage degeneration and pain in the rat meniscal tear model of post-traumatic arthritis. In this report, we show that after a single IA injection to naïve rats and rats that underwent unilateral meniscal tear, [(125)I]LUB:1 had a tri-phasic disposition profile, with the alpha, beta, and gamma half-life estimates of 4.5 h, 1.5 days, and 2.1 weeks, respectively. We hypothesize that the terminal phase kinetics was related to [(125)I]LUB:1 binding to its ligands. [(125)I]LUB:1 was detected on articular cartilage surfaces as long as 28 days after single IA injection. Micro-autoradiography analysis suggested that [(125)I]LUB:1 tended to localize to damaged joint surfaces in rats with meniscal tear. After a single intravenous (IV) dose to rats, [(125)I]LUB:1 was eliminated rapidly from the systemic circulation, with a mean total body clearance of 154 mL/h/kg and a mean elimination half-life (t (1/2)) of 6.7 h. Overall, LUB:1 has met a desired disposition profile of a potential therapeutic intended for an IA administration: target tissue (knee) retention and fast elimination from the systemic circulation after a single IA or IV dose.

LXR modulation blocks prostaglandin E2 production and matrix degradation in cartilage and alleviates pain in a rat osteoarthritis model.

Osteoarthritis (OA), the most common arthritic condition in humans, is characterized by the progressive degeneration of articular cartilage accompanied by chronic joint pain. Inflammatory mediators, such as cytokines and prostaglandin E(2) (PGE(2)) that are elevated in OA joints, play important roles in the progression of cartilage degradation and pain-associated nociceptor sensitivity. We have found that the nuclear receptor family transcription factors Liver X Receptors (LXRalpha and -beta) are expressed in cartilage, with LXRbeta being the predominant isoform. Here we show that genetic disruption of Lxrbeta gene expression in mice results in significantly increased proteoglycan (aggrecan) degradation and PGE(2) production in articular cartilage treated with IL-1beta, indicating a protective role of LXRbeta in cartilage. Using human cartilage explants, we found that activation of LXRs by the synthetic ligand GW3965 significantly reduced cytokine-induced degradation and loss of aggrecan from the tissue. Furthermore, LXR activation dramatically inhibited cytokine-induced PGE(2) production by human osteoarthritic cartilage as well as by a synovial sarcoma cell line. These effects were achieved at least partly by repression of the expression of ADAMTS4, a physiological cartilage aggrecanase, and of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, key enzymes in the PGE(2) synthesis pathway. Consistent with our in vitro observations, oral administration of GW3965 potently alleviated joint pain in a rat meniscal tear model of osteoarthritis.

Prevention of cartilage degeneration in a rat model of osteoarthritis by intraarticular treatment with recombinant lubricin.

OBJECTIVE: Lubricin, also referred to as superficial zone protein and PRG4, is a synovial glycoprotein that supplies a friction-resistant, antiadhesive coating to the surfaces of articular cartilage, thereby protecting against arthritis-associated tissue wear and degradation. This study was undertaken to generate and characterize a novel recombinant lubricin protein construct, LUB:1, and to evaluate its therapeutic efficacy following intraarticular delivery in a rat model of osteoarthritis (OA). METHODS: Binding and localization of LUB:1 to cartilage surfaces was assessed by immunohistochemistry. The cartilage-lubricating properties of LUB:1 were determined using a custom friction testing apparatus. A cell-binding assay was performed to quantify the ability of LUB:1 to prevent cell adhesion. Efficacy studies were conducted in a rat meniscal tear model of OA. One week after the surgical induction of OA, LUB:1 or phosphate buffered saline vehicle was administered by intraarticular injection for 4 weeks, with dosing intervals of either once per week or 3 times per week. OA pathology scores were determined by histologic analysis. RESULTS: LUB:1 was shown to bind effectively to cartilage surfaces, and facilitated both cartilage boundary lubrication and inhibition of synovial cell adhesion. Treatment of rat knee joints with LUB:1 resulted in significant disease-modifying, chondroprotective effects during the progression of OA, by markedly reducing cartilage degeneration and structural damage. CONCLUSION: Our findings demonstrate the potential use of recombinant lubricin molecules in novel biotherapeutic approaches to the treatment of OA and associated cartilage abnormalities.

Melatonin-depleted blood from premenopausal women exposed to light at night stimulates growth of human breast cancer xenografts in nude rats.

The increased breast cancer risk in female night shift workers has been postulated to result from the suppression of pineal melatonin production by exposure to light at night. Exposure of rats bearing rat hepatomas or human breast cancer xenografts to increasing intensities of white fluorescent light during each 12-hour dark phase (0-345 microW/cm2) resulted in a dose-dependent suppression of nocturnal melatonin blood levels and a stimulation of tumor growth and linoleic acid uptake/metabolism to the mitogenic molecule 13-hydroxyoctadecadienoic acid. Venous blood samples were collected from healthy, premenopausal female volunteers during either the daytime, nighttime, or nighttime following 90 minutes of ocular bright, white fluorescent light exposure at 580 microW/cm2 (i.e., 2,800 lx). Compared with tumors perfused with daytime-collected melatonin-deficient blood, human breast cancer xenografts and rat hepatomas perfused in situ, with nocturnal, physiologically melatonin-rich blood collected during the night, exhibited markedly suppressed proliferative activity and linoleic acid uptake/metabolism. Tumors perfused with melatonin-deficient blood collected following ocular exposure to light at night exhibited the daytime pattern of high tumor proliferative activity. These results are the first to show that the tumor growth response to exposure to light during darkness is intensity dependent and that the human nocturnal, circadian melatonin signal not only inhibits human breast cancer growth but that this effect is extinguished by short-term ocular exposure to bright, white light at night. These mechanistic studies are the first to provide a rational biological explanation for the increased breast cancer risk in female night shift workers.

Melatonin desensitizes endogenous MT2 melatonin receptors in the rat suprachiasmatic nucleus: relevance for defining the periods of sensitivity of the mammalian circadian clock to melatonin.

The hormone melatonin phase shifts circadian rhythms generated by the mammalian biological clock, the suprachiasmatic nucleus (SCN) of the hypothalamus, through activation of G protein-coupled MT2 melatonin receptors. This study demonstrated that pretreatment with physiological concentrations of melatonin (30-300 pM or 7-70 pg/mL) decreased the number of hMT2 melatonin receptors heterologously expressed in mammalian cells in a time and concentration-dependent manner. Furthermore, hMT2-GFP melatonin receptors heterologously expressed in immortalized SCN2.2 cells or in non-neuronal mammalian cells were internalized upon pretreatment with both physiological (300 pM or 70 pg/mL) and supraphysiological (10 nM or 2.3 ng/mL) concentrations of melatonin. The decrease in MT2 melatonin receptor number induced by melatonin (300 pM for 1 h) was reversible and reached almost full recovery after 8 h; however, after treatment with 10 nM melatonin full recovery was not attained even after 24 h. This recovery process was partially protein synthesis dependent. Furthermore, exposure to physiological concentrations of melatonin (300 pM) for a time mimicking the nocturnal surge (8 h) desensitized functional responses mediated through melatonin activation of endogenous MT2 receptors, i.e., stimulation of protein kinase C (PKC) in immortalized SCN2.2 cells and phase shifts of circadian rhythms of neuronal firing in the rat SCN brain slice. We conclude that in vivo the nightly secretion of melatonin desensitizes endogenous MT2 melatonin receptors in the mammalian SCN thereby providing a temporally integrated profile of sensitivity of the mammalian biological clock to a melatonin signal.

Immortalized cells from the rat suprachiasmatic nucleus express functional melatonin receptors.

Immortalized SCN2.2 cells retain most biochemical and biophysical characteristics of the native rat SCN including the expression of clock genes and circadian regulatory proteins, and its distinctive pacemaker function. This study assessed the expression and signaling of MT(1) and MT(2) melatonin receptors in SCN2.2 cells. SCN2.2 cells express MT(1) and MT(2) receptors mRNA as detected by RT-PCR. In situ hybridization with digoxigenin-labeled probes demonstrated that mRNA for MT(1) and MT(2) melatonin receptors is expressed mostly in cells with neuronal-like morphology, representing 10.8+/-2.2% and 9.8+/-0.2%, respectively, of the SCN2.2 cell population. MT(1) and MT(2) melatonin receptor proteins are expressed in both rat SCN2.2 cells and rat SCN tissue as demonstrated by Western blot analysis with specific receptor antiserum. Melatonin (0.1-100 nM) inhibited forskolin (20 microM)-stimulated cAMP formation in a dose-dependent manner and this effect was blocked by the competitive melatonin receptor antagonist luzindole (100-1000 nM). Furthermore, melatonin (1 nM) stimulated protein kinase C (PKC) activity by approximately 2-fold. The selective MT(2) receptor antagonist 4P-PDOT (100 nM) blocked this effect, indicating that the melatonin-mediated increase in PKC activity occurs through activation of MT(2) melatonin receptors. We conclude that SCN2.2 cells express functional melatonin receptors, providing an in vitro model to unveil the melatonin signaling pathway(s) involved in the regulation of circadian rhythms.

Molecular pharmacology, regulation and function of mammalian melatonin receptors.

Melatonin (5-methoxy-N-acetyltryptamine), dubbed the hormone of darkness, is released following a circadian rhythm with high levels at night. It provides circadian and seasonal timing cues through activation of G protein-coupled receptors (GPCRs) in target tissues (1). The discovery of selective melatonin receptor ligands and the creation of mice with targeted disruption of melatonin receptor genes are valuable tools to investigate the localization and functional roles of the receptors in native systems. Here we describe the pharmacological characteristics of melatonin receptor ligands and their various efficacies (agonist, antagonist, or inverse agonist), which can vary depending on tissue and cellular milieu. We also review melatonin-mediated responses through activation of melatonin receptors (MT1, MT2, and MT3) highlighting their involvement in modulation of CNS, hypothalamic-hypophyseal-gonadal axis, cardiovascular, and immune functions. For example, activation of the MT1 melatonin receptor inhibits neuronal firing rate in the suprachiasmatic nucleus (SCN) and prolactin secretion from the pars tuberalis and induces vasoconstriction. Activation of the MT2 melatonin receptor phase shifts circadian rhythms generated within the SCN, inhibits dopamine release in the retina, induces vasodilation, enhances splenocyte proliferation and inhibits leukocyte rolling in the microvasculature. Activation of the MT3 melatonin receptor reduces intraocular pressure and inhibits leukotriene B4-induced leukocyte adhesion. We conclude that an accurate characterization of melatonin receptors mediating specific functions in native tissues can only be made using receptor specific ligands, with the understanding that receptor ligands may change efficacy in both native tissues and heterologous expression systems.

Regulation of basal rhythmicity in protein kinase C activity by melatonin in immortalized rat suprachiasmatic nucleus cells.

Melatonin phase shifts circadian rhythms of the neuronal firing rate and stimulates PKC activity at dusk (CT 10) and dawn (CT 23) in the rat suprachiasmatic nucleus (SCN) slice via activation of the MT(2) melatonin receptor. We demonstrated that in the SCN2.2 cells basal PKC activity follows a rhythmic oscillation with an acrophase during the subjective dark phase (CT 14-CT 22) and nadirs during the subjective light phase at CT 2 and CT 10. Melatonin (0.01-10 nM, 10 min) significantly doubled basal PKC activity at CT 2 and CT 10, and decreased basal PKC activity at CT 6. We conclude that melatonin regulates the basal rhythm in PKC activity generated in SCN2.2 cells at the same periods of sensitivity observed in the native SCN.

1,25-Dihydroxyvitamin D3 selectively translocates PKCalpha to nuclei in ROS 17/2.8 cells.

We have investigated protein kinase C (PKC) regulation by 1,25-(OH)2D3 in the rat osteosarcoma cell line ROS 17/2.8 since previous reports have implicated PKC in the 1,25-(OH)2D3-mediated regulation of osteocalcin gene expression (J. Biol. Chem. 267 (1992) 12562; Endocrinology 136 (1995) 5685). Here we report that 1,25-(OH)2D3 increased PKCalpha, but not PKCbetaI, epsilon or zeta, levels in the nuclear fraction in a time-dependent manner. Unlike PMA, 1,25-(OH)2D3 did not alter the association of any of the expressed PKC isoenzymes with the plasma membrane. Treatment with 20 nM 1,25-(OH)2D3 for 15 min, 30 min, 1 h and 24 h increased PKCalpha levels in the nuclear fraction by 2.3- to 2.6-fold. Nuclear PKCalpha expression was also increased with doses of 1,25-(OH)2D3 as low as a 0.05 nM. 1,25-(OH)2D3-mediated stabilization of osteocalcin mRNA (Arch. Biochem. Biophys. 332 (1996) 142) was inhibited with bisindolylmaleimide treatment, suggesting that PKCalpha may be involved in the 1,25-(OH)2D3-mediated regulation of osteocalcin gene expression.