Dopaminergic systems are thought to mediate the rewarding and reinforcing effects of palatable food. However, the relative contribution of different dopamine receptor subtypes is not clear. We used dopamine D1 receptor deficient mice (D1 -/-) and their wild-type and heterozygous littermates to study the role of the D1 receptor in palatable food reinforced behaviour using operant responding and free access paradigms. Non-deprived mice were trained to press a lever for sucrose pellets under three schedules of reinforcement including fixed ratios (FR-1 and FR-4) and a progressive ratio (PR). Responding on one lever was reinforced by the delivery of a sucrose pellet or solution while responding on a second lever had no programmed consequences. Initially, D1 mutant mice took longer to learn to discriminate between the two levers and had significantly lower operant responding for sucrose pellets and solution than wild-type and heterozygous mice under all schedules of reinforcement. Food deprivation enhanced responding on the active lever in all mice although it remained significantly lower in D1 -/- mice than in control mice. Following extinction of sucrose reinforcement and reversal of the levers, D1 -/- mice showed deficits in extinguishing and reversing previously learned responses. Home cage intake and preference of sucrose pellets and solutions when given under free-choice access paradigms were similar among the groups. These results suggest that the dopamine D1 receptor plays a role in the motivation to work for reward (palatable food) but not in reward perception and is critical in learning new but relevant information and discontinuing previously learned responses.

We have investigated the role of protein kinase C (PKC) signal transduction pathways in parathyroid hormone (PTH) regulation of insulin-like growth factor-binding protein-5 (IGFBP-5) gene expression in the rat osteoblast-like cell line UMR-106–01. Involvement of the PKC pathway was determined by the findings that bisindolylmaleimide I inhibited 40% of the PTH effect, and 1 μM bovine PTH-(3–34) stimulated a 10-fold induction of IGFBP-5 mRNA. PTH-(1–34) and PTH-(3–34) (100 nM) both stimulated PKC-δ translocation from the membrane to the nuclear fraction. Rottlerin, a PKC-δ-specific inhibitor, and a dominant negative mutant of PKC-δ were both able to significantly inhibit PTH-(1–34) and PTH-(3–34) induction of IGFBP-5 mRNA, suggesting a stimulatory role for PKC-δ in the effects of PTH. Phorbol 12-myristate 13-acetate (PMA) stimulated PKC-α translocation from the cytosol to the membrane and inhibited ∼50% of the PTH-(1–34), forskolin, and 8-bromoadenosine 3′,5′-cyclic monophosphate-stimulated IGFBP-5 mRNA levels, suggesting that PKC-α negatively regulates protein kinase A (PKA)-mediated induction of IGFBP-5 mRNA. These results suggest that the induction of IGFBP-5 by PTH is both PKA and PKC dependent and PKC-δ is the primary mediator of the effects of PTH via the PKC pathway.

In osteoblasts parathyroid hormone (PTH) stimulates the PTH/PTH-related peptide (PTHrP) receptor (PTH1R) that couples via G(s) to adenylyl cyclase stimulation and via G(11) to phospholipase C (PLC) stimulation. We have investigated the effect of increasing G(11)alpha levels in UMR 106-01 osteoblastic cells by transient transfection with cDNA encoding G(11)alpha on PTH stimulation of PLC and protein kinase C (PKC) as well as PTH regulation of mRNA encoding matrix metalloproteinase-13 (MMP-13). Transfection with G(11)alpha cDNA resulted in a 5-fold increase in PTH-stimulated PLC activity with no change in PTH-stimulated adenylyl cyclase. PTH-induced translocation of PKC-betaI, -delta, and -zeta to the cell membrane and PKC-zeta to the nucleus was also increased. Increased G(11)alpha protein resulted in increased stimulation of MMP-13 mRNA levels at all doses of PTH. There was a 2.5 +/- 0.35 fold increase in maximal PTH-stimulation of c-jun mRNA and smaller but significant increases in c-fos accompanied by increased basal and PTH-stimulated AP-1 binding in cells expressing increased G(11)alpha. Runx-2 mRNA and protein levels were not significantly increased by increased G(11)alpha expression. The increase in PTH stimulation of c-jun, c-fos, and MMP-13 in G(11)alpha-transfected cells were all blocked by bisindolylmaleimide I, a selective inhibitor of PKC. These results demonstrate that regulation of the PLC pathway through the PTH1R is significantly increased by elevating expression of G(11)alpha in osteoblastic cells. This leads to increased PTH stimulation of MMP-13 expression by increased stimulation of AP-1 factors c-jun and c-fos.

We have previously shown that parathyroid hormone (PTH) stimulates the expression of insulin-like growth factor binding protein-5 (IGFBP-5) transcript levels in the osteosarcoma cell-line, UMR106-01 cells. In the present study we examined the molecular basis for the PTH induction of IGFBP-5 mRNA in these cells. PTH had no effect on the half-life of the IGFBP-5 transcript but did stimulate the transactivation of the proximal 889 base pairs of the rat IGFBP 58 flanking region in a luciferase fusion construct, suggesting that PTH stimulates transcript levels through transcriptional mechanisms. Progressive 58 deletions to -59 base pairs of the proximal promoter region had no effect on PTH induction of transactivation, indicating that an element existed within the first -59 base pairs upstream of the transcription start site that was responsive to PTH. Within the -59 base pairs there are CCAAT/enhancer binding protein (C/EBP), E-box, nuclear factor-1 (NF-1) and activator protein-2 (AP-2) elements. Mutation of the C/EBP, E-box or NF-1 elements had no effect on the ability of PTH to induce the transactivation of the IGFBP-5 promoter. Mutation of the AP-2 element resulted in a 40% reduction of PTH-stimulated luciferase activity. When three tandem repeats of the AP-2 consensus sequence were fused to a luciferase reporter, PTH stimulated a 25% increase in reporter activity. Electrophoretic mobility shift assays using UMR106-01 cell nuclear extracts showed that PTH caused a prominent shifted band in a probe spanning the region containing all four elements. The shifted band was almost completely absent when the probe contained a mutated AP-2 element. These results suggest that the AP-2 element functions in the PTH induction of IGFBP-5 gene expression.