Notch signaling in postnatal joint chondrocytes, but not subchondral osteoblasts, is required for articular cartilage and joint maintenance.

OBJECTIVE: Notch signaling has been identified as a critical regulator in cartilage development and joint maintenance, and loss of Notch signaling in all joint tissues results in an early and progressive osteoarthritis (OA)-like pathology. This study investigated the targeted cell population within the knee joint in which Notch signaling is required for normal cartilage and joint integrity. METHODS: Two loss-of-function mouse models were generated with tissue-specific knockout of the core Notch signaling component, RBPjκ. The AcanCre(ERT2) transgene specifically removed Rbpjκ floxed alleles in postnatal joint chondrocytes, while the Col1Cre(2.3kb) transgene deleted Rbpjκ in osteoblast populations, including subchondral osteoblasts. Mutant and control mice were analyzed via histology, immunohistochemistry (IHC), real-time quantitative polymerase chain reaction (qPCR), X-ray, and microCT imaging at multiple time-points. RESULTS: Loss of Notch signaling in postnatal joint chondrocytes results in a progressive OA-like pathology, and triggered the recruitment of non-targeted fibrotic cells into the articular cartilage potentially due to mis-regulated chemokine expression from within the cartilage. Upon recruitment, these fibrotic cells produced degenerative enzymes that may lead to the observed cartilage degradation and contribute to a significant portion of the age-related OA-like pathology. On the contrary, loss of Notch signaling in subchondral osteoblasts did not affect normal cartilage development or joint maintenance. CONCLUSIONS: RBPjκ-dependent Notch signaling in postnatal joint chondrocytes, but not subchondral osteoblasts, is required for articular cartilage and joint maintenance.

Smad1 plays an essential role in bone development and postnatal bone formation.

OBJECTIVES: To determine the role of Smad1 in bone development and postnatal bone formation. METHODS: Col2a1-Cre transgenic mice were bred with Smad1(fx/fx) mice to produce chondrocyte-specific Smad1 conditional knockout (cKO) mice. Embryonic skeletal preparation and staining were performed, alkaline phosphatase activity (ALP) and relative gene expression were examined in isolated primary cells. Smad1(fx/fx) mice were also bred with Col1a1-Cre transgenic mice to produce osteoblast-specific Smad1 cKO mice. Postnatal bone formation was assessed by micro-computed tomography (µCT) and histological analyses in 2-month-old mice. Mineralized bone nodule formation assay, 5-bromo-2'-deoxy-uridine (BrdU) labeling and gene expression analysis were performed. RESULTS: Mice with chondrocyte- and osteoblast-specific deletion of the Smad1 gene are viable and fertile. Calvarial bone development was delayed in chondrocyte-specific Smad1 cKO mice. In osteoblast-specific Smad1 cKO mice, BMP signaling was partially inhibited and mice developed an osteopenic phenotype. Osteoblast proliferation and differentiation were impaired in osteoblast-specific Smad1 cKO mice. CONCLUSIONS: Smad1 plays an essential role in bone development and postnatal bone formation.

Overexpression of the alpha1B-adrenergic receptor causes apoptotic neurodegeneration: multiple system atrophy.

Progress toward elucidating the function of alpha1B-adrenergic receptors (alpha1BARs) in the central nervous system has been constrained by a lack of agonists and antagonists with adequate alpha1B-specificity. We have obviated this constraint by generating transgenic mice engineered to overexpress either wild-type or constitutively active alpha1BARs in tissues that normally express the receptor, including the brain. All transgenic lines showed granulovacular neurodegeneration, beginning in alpha1B-expressing domains of the brain and progressing with age to encompass all areas. The degeneration was apoptotic and did not occur in non-transgenic mice. Correspondingly, transgenic mice showed an age-progressive hindlimb disorder that was parkinsonian-like, as demonstrated by rescue of the dysfunction by 3, 4-dihydroxyphenylalanine and considerable dopaminergic-neuronal degeneration in the substantia nigra. Transgenic mice also had a grand mal seizure disorder accompanied by a corresponding dysplasia and neurodegeneration of the cerebral cortex. Both behavioral phenotypes (locomotor impairment and seizure) could be partially rescued with the alpha1AR antagonist terazosin, indicating that alpha1AR signaling participated directly in the pathology. Our results indicate that overstimulation of alpha1BAR leads to apoptotic neurodegeneration with a corresponding multiple system atrophy indicative of Shy-Drager syndrome, a disease whose etiology is unknown.

Activation of phosphoinositide metabolism by parathyroid hormone in growth plate chondrocytes.

Parathyroid hormone (PTH) is one of the most potent stimulators of growth plate chondrocyte mitogenesis that has been reported. However, study of the second messenger signaling mechanisms involved in the transduction of the hormone's effects on these cells is incomplete. Our data indicate that in addition to stimulating cyclic adenosine-3'5'-monophosphate metabolism, PTH also activates the phosphoinositide cascade, the pathway responsible for the generation of inositol-1,4,5-trisphosphate dependent Ca2+ signals. Our conclusion that PTH activates the phosphoinositide cascade is based on data that demonstrate: (1) the Ca2+ transients evoked by the hormone are dependent on intracellular Ca2+ stores; (2) the hormone stimulates the release of radiolabeled inositol from GPC plasma membranes; and (3) the hormone stimulates a greater than 8-fold increase in cytosolic inositol-1,4,5-trisphosphate pool size.

Cytosolic free calcium concentrations in avian growth plate chondrocytes.

Isolated avian growth plate chondrocytes convert the acetoxymethyl ester (AM) form of Fura-2 quickly and efficiently to the Ca2(+)-sensitive pentacarboxylic acid (FA) form. Control experiments indicate that the Kd for intracellular Fura-2/FA is very close to that of extracellular Fura-2/FA at the same ionic strength and pH and that the Fura-2/FA fluorescence from indicator converted by intracellular organelles is quite small. Correcting for the effects of extracellular Fura-2/FA and partial hydrolysis products has improved the accuracy of determination of intracellular [Ca2+] over earlier measurements in chondrocytes. Cytosolic [Ca2+] in isolated growth plate chondrocytes (containing cells from each maturational stage) is found to require approximately 9 hours to recover from the isolation process. After this recovery period, cytosolic [Ca2+] in these cells converges to approximately 70 nM regardless of the [Ca2+] of the recovery medium, suggesting regulation of cytosolic [Ca2+] to a set point. Chondrocytes that are separated into maturationally distinct fractions using countercurrent centrifugal elutriation show an increase in cytosolic [Ca2+] with cellular maturation. The least mature resting cells have a [Ca2+] near 57 nM, while the most mature hypertrophic cells are around 95 nM.

Runx2-mediated activation of the Bax gene increases osteosarcoma cell sensitivity to apoptosis.

The Runx family of transcription factors regulate cell growth and differentiation, and control the expression of target genes involved in cell fate decisions. We examined the role of the bone-related member of this family, Runx2, in regulating apoptosis via modulation of the Bcl2 family of genes in the osteosarcoma cell line Saos2. Our data demonstrate that Runx2 directly binds to two Runx-specific regulatory elements on the human bax promoter thereby inducing Bax expression. Furthermore, bone morphogenetic protein-induced or vector-mediated expression of Runx2 resulted in upregulation of Bax expression, and subsequent increased sensitivity of Saos2 cells to apoptosis. Finally, the observed upregulation of Bax expression and increased apoptosis were Runx2 dependent as Runx2 loss of function abrogated these effects. Our study provides the first evidence for Bax as a direct target of Runx2, suggesting that Runx2 may act as a proapoptotic factor in osteosarcoma cells.

The Na(+)-independent Ca2+ efflux mechanism of liver mitochondria is not a passive Ca2+/2H+ exchanger.

Whether the Na(2+)-independent Ca2+ efflux mechanism of liver mitochondria is a Ca2+/2H+ exchanger and whether this exchanger is a passive mechanism have been controversial since shortly after the discovery of this mechanism. Here, a new approach to determining if the mechanism is passive is developed based on the energy available to a passive Ca2+/2H+ exchanger. Conditions are identified in which the Na(+)-independent Ca2+ efflux mechanism transports Ca2+ out of mitochondria against a Ca2+ gradient many times greater than that possible for a passive Ca2+/2H+ exchanger, thus ruling this out as a possible mechanism.

Cloning, cell-type specificity, and regulatory function of the mouse alpha(1B)-adrenergic receptor promoter.

The functionality of a 3422-base pair promoter fragment from the mouse alpha(1B)-adrenergic receptor (alpha(1B)AR) gene was examined. This fragment, cloned from a mouse genomic library, was found to have significant sequence homology to the known human and rat alpha(1B)AR promoters. However, the consensus motif of several key cis-acting elements is not conserved among the rat, human, and mouse genes, suggesting species specificity. Confirming fidelity of the murine promoter, robust in vitro expression of a chloramphenicol acetyltransferase (CAT) reporter was detected in known alpha(1B)AR-expressing BC(3)H1, NB41A3, and DDT(1)MF-2 cells transiently transfected with a promoter-CAT construct. Conversely, minimal CAT expression was detected in known alpha(1B)AR-negative RAT-1 and R3T3 cells. These findings were extended by transfecting the same promoter-CAT construct into various primary cell types. In support of the hypothesis that alpha(1)ARs are differentially expressed in the smooth muscle of the vasculature, primary cultures of superior mesenteric and renal artery vascular smooth muscle cells showed significantly stronger CAT expression than did vascular smooth muscle cells derived from pulmonary, femoral, and iliac arteries. Primary osteoblastic bone-forming cells, which are known to be alpha(1B)AR negative, showed minimal CAT expression. Indicating regulatory function through cis-acting elements, RAT-1, R3T3, NB41A3, BC(3)H1, and DDT(1)MF2 cells transfected with the promoter-CAT construct all showed increased CAT production when challenged with forskolin or hypoxic conditions. Additionally, tissue-specific regulation of the promoter was observed when cells were simultaneously challenged with both forskolin and hypoxia. These results collectively demonstrate that a 3.4-kb PvuII fragment of the murine alpha(1B)AR gene promoter can: 1) drive tissue-specific production of a CAT reporter in both clonal and primary cell lines; and 2) confer tissue-specific regulation of that CAT reporter when induced by challenge with forskolin and/or hypoxic conditions.

Characterization of voltage-sensitive calcium channels in growth plate chondrocytes.

Growth plate chondrocytes (GPCs), cells integrally involved with the process of endochondral bone formation, facilitate Ca2+ infux to provide a source of ion for processes such as Ca2+ signaling and matrix vesicle loading. We hypothesize that this Ca2+ entry into GPCs is achieved through the action of voltage-sensitive Ca2+ channels. This hypothesis was tested by measuring intracellular [Ca2+] changes in fura 2-loaded GPCs that were depolarized by challenge with a K(+)-containing medium. KCl doses between 55 and 95 mM evoked significant Ca2+ responses that were blocked by addition of extracellular EGTA. The Ca2+ response evoked by 95 mM K+ was insensitive to 100 microM doses of nifedipine or nitrendipine, ruling out L-type channel involvement. This finding was corroborated by the observation that 10 microM BAY K 8644 did not activate a Ca2+ response of its own. However, 10 microM Cd2+ significantly inhibited the 95 mM Ks(+)-evoked effects, suggesting N-type channel activity. Use of 1 microM Ni+ in an attempt to block possible T-type channel activity caused nonspecific cellular effects, precluding pharmacological assessment of a possible T-type channel activity. These data (i) provide the first direct evidence for voltage-sensitive Ca2+ channel activity in GPCs and (ii) suggest at least partial facilitation of that activity through N type channels.

Novel aromatic residues in transmembrane domains IV and V involved in agonist binding at alpha(1a)-adrenergic receptors.

We examined the role that aromatic residues located in the transmembrane helices of the alpha(1a)-adrenergic receptor play in promoting antagonist binding. Since alpha(1)-antagonists display low affinity binding at beta(2)-adrenergic receptors, two phenylalanine residues, Phe-163 and Phe-187, of the alpha(1a)-AR were mutated to the corresponding beta(2)-residue. Neither F163Q nor F187A mutations of the alpha(1a) had any effect on the affinity of the alpha(1)-antagonists. However, the affinity of the endogenous agonist epinephrine was reduced 12.5- and 8-fold by the F163Q and F187A mutations, respectively. An additive loss in affinity (150-fold) for epinephrine was observed at an alpha(1a) containing both mutations. The loss of agonist affinity scenario could be reversed by a gain of affinity with mutation of the corresponding residues in the beta(2) to the phenylalanine residues in the alpha(1a). We propose that both Phe-163 and Phe-187 are involved in independent aromatic interactions with the catechol ring of agonists. The potency but not the efficacy of epinephrine in stimulating phosphatidylinositol hydrolysis was reduced 35-fold at the F163Q/F187A alpha(1a) relative to the wild type receptor. Therefore, Phe-163 and Phe-187 represent novel binding contacts in the agonist binding pocket of the alpha(1a)-AR, but are not involved directly in receptor activation.

Identification of a conserved switch residue responsible for selective constitutive activation of the beta2-adrenergic receptor.

A cysteine-to-phenylalanine mutation of residue 116 in the third transmembrane domain of the beta2-adrenergic receptor caused selective constitutive activation of Na+/H+ exchange through a pathway not involving cAMP. This selectivity was identified by comparing binding and signaling characteristics of wild-type (WT) versus C116F mutant receptors transiently transfected into COS-1 cells. Indicating constitutive activity, ligand binding to the C116F mutant showed a 78-fold higher than WT affinity for isoproterenol and a 40-fold lower than WT affinity for ICI 118551. Although agonist-independent activation of cAMP production was not exhibited by the C116F mutant, a constitutive stimulation of the Na+/H+ exchanger (NHE1) was observed. This was identified by measuring either basal intracellular pH (pHi) or rate of pHi recovery from cellular acid load. Due to a higher rate of H+ efflux through NHE1, C116F transfectants exhibited a significantly higher pHi (7.42) than did WT transfectants (7.1). Furthermore, the rate of pHi recovery from acid load facilitated by NHE1 was 2.1-fold faster in mutant transfectants than in WT transfectants. The lower rate seen in the WT case was stimulated by epinephrine, and the higher rate seen in the mutant case was inhibited by ICI 118551. These findings, which show that a C116F mutation of the beta2-adrenergic receptor evokes selective constitutive coupling to NHE1 over cAMP, form the basis of our prediction that multiple and distinct activation states can exist in G protein-coupled receptors.

Cyclic-AMP-dependent protein kinase activity is not required by parathyroid hormone to stimulate phosphoinositide signaling in chondrocytes but is required to transduce the hormone's proliferative effect.

Parathyroid hormone (PTH), an activator of both cAMP and phosphoinositide (PI) signaling in growth plate chondrocytes (GPCs), is generally believed to trigger each of these pathways through interactions with separate G proteins. Recently, however, activation of cAMP-dependent protein kinase (pkA) has been found to cause a stimulation of the PI cascade in hepatocytes. This finding raises the possibility that PTH stimulation of PI metabolism in GPCs may really be a secondary event, mediated through a primary stimulation of pkA. Experiments discussed in the present report indicate that the PTH stimulation of PI metabolism in GPCs is independent of pkA activity. The data show that (1) unlike the Ca2+ response evoked by PTH, the responses evoked by dibutyryl-cAMP or Sp diastereomer of cyclic adenosine-3',5'-monophosphothioate, two activators of pkA, require an extracellular Ca2+ source; (2) also unlike PTH, activation of pkA by these same cAMP analogs does not cause an increase in cellular inositol-1,4,5-trisphosphate; and (3) specific inhibition of pkA with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinsulfanomide (H-89) or Rp diastereomer of cyclic adenosine-3',5'-monophosphothioate (Rp cAMPS) has no effect on the ability of PTH to evoke its normal Ca2+ response. Furthermore, data presented indicate that the PTH stimulation of GPC proliferation does not require Ca2+ signals, but rather is at least partially dependent on pkA. The data show that either loading the cells with the Ca2+ buffer bis-(o-aminophenoxy)ethane-N,N,N',N'-tetracetic acid or depleting the cells of intracellularly stored Ca2+ is without effect on the stimulation of DNA synthesis by the hormone. Inhibition of pkA activity with H-89 or Rp-cAMPS, in contrast, leads to a significant reduction in the ability of PTH to stimulate its proliferative effect.

Identification of functional insulin-like growth factor-II/mannose-6-phosphate receptors in isolated bone cells.

The role of the IGF-II/cation independent mannose-6-phosphate (IGF-II/M6P) receptor in the transduction of cellular effects evoked by IGF-II has been extensively debated in the literature. Many reports suggest that IGF-II transduces its effects through the IGF-I receptor, while others show that IGF-II utilizes the type II receptor to affect cellular activity. This study 1) verifies the presence of the IGF-II/M6P receptor in rat calvarial osteoblasts, and 2) evaluates the ability of the receptor to initiate intracellular signals. Using conventional receptor binding assays, it was found that osteoblasts bind IGF-II with high affinity. Scatchard analyses indicated that there are 5.08 x 10(4) IGF-II/M6P receptors per osteoblast with a Kd near 2.0 nM). The receptor protein was further identified by cross-linking with 125I-IGF-II. Northern analysis was used to identify an mRNA transcript for the IGF-II/M6P receptor protein. To examine if the IGF-II/M6P receptor can initiate second messenger signals, the ability of IGF-II to evoke Ca2+ transients was evaluated. Osteoblasts pretreated with IGF-I did not lose their ability to respond to IGF-II. Further, a polyclonal antibody against the rat IGF-II/M6P receptor (R-II-PAB1) 1) was able to evoke its own Ca2+ response, and 2) was able to block the generation of Ca2+ transients caused by IGF-II. The data in this report show that the osteoblastic Ca2+ response to IGF-II appears to be caused by an intracellular release of Ca2+ which is mediated by the IGF-II/M6P receptor making it possible to envision how the receptor may be an important modulator of osteoblast mediated osteogenesis.

Phe-308 and Phe-312 in transmembrane domain 7 are major sites of alpha 1-adrenergic receptor antagonist binding. Imidazoline agonists bind like antagonists.

Although agonist binding in adrenergic receptors is fairly well understood and involves residues located in transmembrane domains 3 through 6, there are few residues reported that are involved in antagonist binding. In fact, a major docking site for antagonists has never been reported in any G-protein coupled receptor. It has been speculated that antagonist binding is quite diverse depending upon the chemical structure of the antagonist, which can be quite different from agonists. We now report the identification of two phenylalanine residues in transmembrane domain 7 of the alpha(1a)-adrenergic receptor (Phe-312 and Phe-308) that are a major site of antagonist affinity. Mutation of either Phe-308 or Phe-312 resulted in significant losses of affinity (4-1200-fold) for the antagonists prazosin, WB4101, BMY7378, (+) niguldipine, and 5-methylurapidil, with no changes in affinity for phenethylamine-type agonists such as epinephrine, methoxamine, or phenylephrine. Interestingly, both residues are involved in the binding of all imidazoline-type agonists such as oxymetazoline, cirazoline, and clonidine, confirming previous evidence that this class of ligand binds differently than phenethylamine-type agonists and may be more antagonist-like, which may explain their partial agonist properties. In modeling these interactions with previous mutagenesis studies and using the current backbone structure of rhodopsin, we conclude that antagonist binding is docked higher in the pocket closer to the extracellular surface than agonist binding and appears skewed toward transmembrane domain 7.

Hypotension, autonomic failure, and cardiac hypertrophy in transgenic mice overexpressing the alpha 1B-adrenergic receptor.

alpha(1)-Adrenergic receptors (alpha(1A), alpha(1B), and alpha(1D)) are regulators of systemic arterial blood pressure and blood flow. Whereas vasoconstrictory action of the alpha(1A) and alpha(1D) subtypes is thought to be mainly responsible for this activity, the role of the alpha(1B)-adrenergic receptor (alpha(1B)AR) in this process is controversial. We have generated transgenic mice that overexpress either wild type or constitutively active alpha(1B)ARs. Transgenic expression was under the control of the isogenic promoter, thus assuring appropriate developmental and tissue-specific expression. Cardiovascular phenotypes displayed by transgenic mice included myocardial hypertrophy and hypotension. Indicative of cardiac hypertrophy, transgenic mice displayed an increased heart to body weight ratio, which was confirmed by the echocardiographic finding of an increased thickness of the interventricular septum and posterior wall. Functional deficits included an increased isovolumetric relaxation time, a decreased heart rate, and cardiac output. Transgenic mice were hypotensive and exhibited a decreased pressor response. Vasoconstrictory regulation by alpha(1B)AR was absent as shown by the lack of phenylephrine-induced contractile differences between ex vivo mesenteric artery preparations. Plasma epinephrine, norepinephrine, and cortisol levels were also reduced in transgenic mice, suggesting a loss of sympathetic nerve activity. Reduced catecholamine levels together with basal hypotension, bradycardia, reproductive problems, and weight loss suggest autonomic failure, a phenotype that is consistent with the multiple system atrophy-like neurodegeneration that has been reported previously in these mice. These results also suggest that this receptor subtype is not involved in the classic vasoconstrictory action of alpha(1)ARs that is important in systemic regulation of blood pressure.