ABSTRACT
We examined the capacity of PTHrP to modulate the terminal differentiation of the preadipocytic cell line, 3T3-L1. These cells express endogenous PTHrP and its receptor, but expression levels were undetectable after differentiation into mature adipocytes. Cells stably overexpressing PTHrP failed to differentiate when induced to undergo adipogenesis and proliferated at a faster rate. MAPK activity was elevated in PTHrP-transfected 3T3-L1 cells, and treatment with the PKA inhibitor H-8 decreased this activity. Inhibition of MAPK kinase with PD098059 permitted terminal differentiation of PTHrP-transfected 3T3-L1 cells to proceed. Although PPAR gamma gene expression levels remained relatively constant in the PTHrP-transfected cells, PPAR gamma phosphorylation was enhanced. Furthermore, the capacity of PPAR gamma to stimulate transcription in the presence of troglitazone was diminished by PTHrP. Expression of the PPAR gamma-regulated adipocyte specific gene aP2 transiently rose and then fell in PTHrP-transfected cells. These results indicate that PTHrP can increase MAPK activity in 3T3-L1 cells via the PKA pathway, thereby enhancing PPAR gamma phosphorylation. This modification can inactivate the transcriptional enhancing activity of PPAR gamma and diminish the expression of adipocyte-specific genes. These studies therefore demonstrate that PTHrP may inhibit the terminal differentiation of preadipocytes and describe a molecular pathway by which this action can be achieved.
Subject(s)
Adipocytes/cytology , Mitogen-Activated Protein Kinases/physiology , Proteins/pharmacology , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolism , 3T3 Cells , Animals , COS Cells , Cell Differentiation/drug effects , Cyclic AMP-Dependent Protein Kinases/physiology , Down-Regulation , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Mice , Parathyroid Hormone-Related Protein , Phosphorylation/drug effects , Proteins/genetics , RNA, Messenger/metabolism , Receptor, Parathyroid Hormone, Type 1 , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Parathyroid Hormone/physiology , Transcription Factors/antagonists & inhibitors , Transcription Factors/physiology , Transcription, Genetic/drug effects , Transcription, Genetic/physiology , TransfectionABSTRACT
PTH-related peptide (PTHrP) has been implicated in a variety of developmental and homeostatic processes. Although mice homozygous for the targeted disruption of the Pthrp gene have greatly expanded our capacity to investigate the developmental roles of the protein, the perinatal lethality of these animals has severely hindered the analysis of Pthrp's postnatal physiological effects. To overcome this obstacle, we have generated mice homozygous for a floxed Pthrp allele, i.e. two loxP sites flanking exon 4 of the Pthrp gene, which encodes most of the protein, with the aim of accomplishing cell type- and tissue-specific deletion of the gene. The ability of the Cre enzyme to cause recombination between the loxP sites and excision of the intervening DNA sequence was tested in vivo by crossing this strain to mice carrying a cre transgene under the transcriptional control of the human beta-actin promoter. The ubiquitous deletion of the floxed allele in the cre/loxP progeny resulted in perinatal lethality as a consequence of aberrant endochondral bone formation, fully recapitulating all the phenotypic abnormalities observed in the conventional Pthrp knockout mouse. The availability of the floxed Pthrp mice will serve as a valuable tool in genetic experiments that aim to investigate the physiological actions of Pthrp in the postnatal state.
Subject(s)
Alleles , Gene Targeting , Proteins/genetics , Viral Proteins , Actins/genetics , Animals , Chromosome Mapping , Humans , Integrases/physiology , Mice , Organ Specificity , Parathyroid Hormone-Related Protein , Recombination, GeneticABSTRACT
Parathyroid hormone-related peptide (PTHrP) has important functions in the control of cellular growth and differentiation. Acting, at least in part, through the PTH-1 receptor, PTHrP profoundly influences chondrocytic and osteogenic cell biology. Studies using knockout and transgenic mouse technology have played a pivotal role in unraveling the physiological role of PTHrP and its receptor in endochondral bone development and adult skeletal homeostasis. Further clarification of these functions will have far-reaching implications in our general understanding of skeletogenesis and the pathophysiology of human skeletal disorders.
