Deletion Of PTHrP Nuclear Localization And Carboxyl Terminus Leads To Skin Aging

Many studies have demonstrated the important role played by the N-terminal region of PTHrP as is the N-terminus of PTH, by interacting with the common PTH/PTHrP receptor; however increasing evidence suggests that PTHrP is a polyhormone with different functions for different domains. The mid-molecular region, amino acids 37-86, is responsible for placental Ca2+ transport and C-terminus 108-139 inhibit osteoclast activity and bone resorption. Previous studies have shown nuclear localization of PTHrP in multiple cell types and have been identified a functional nuclear localization sequence (NLS) in the 87-107 region of the molecule. The intracrine action of PTHrP has been reported in vitro to include inhibition of cell apoptosis and stimulation of cell proliferation. To investigate the function of NLS of PTHrP in vivo, we generate an animal model with disruption of the NLS of PTHrP by introducing a premature termination codon TGA into the PTHrP gene and created a"knock-in"mouse expressing PTHrP (1-84), a form of the protein missing the NLS and its carboxyl terminus (PTHrP KI). Mice homozygous for the knock-in mutation displayed features of impaired growth and early senescence including muscle atrophy, atrophic skin with hyperkeratosis, reduced body fat, and osteoporosis. However, it is unknown what mechanism of PTHrP KI resulted in skin aging.To explore the mechanism of PTHrP KI caused skin aging, the phenotypes of skin were analyzed by the comparison of PTHrP KI mice at postnatal day 1, 7 and 14 to their age-matched wild-type (WT) littermates using histopathologic, cellular and molecular approaches. PTHrP KI mice displayed a premature aging phenotype in skin including thinner skin with hyperkeratosis of the epidermis, reduced collagen and subcutaneous fat deposition in skin matrix. The skin permeability was increased in PTHrP KI mice demonstrated by staining with toluidine blue and the result suggests that PTHrP KI resulted in a faulty permeability barrier. Consequently, deletion of the PTHrP NLS and carboxyl terminus leads to a skin premature senesence.The proliferation of dermatic cells was decreased and the apoptosis of dermatic cells were increased in PTHrP KI mice demonstrated by immunohistochemical staining for the proliferating cell nuclear antigen (PCNA), double staining with Annexin V and propidium iodide and flow Cytometry analysis, respectively. The expression levels of cell cycle protein D1 and E and cyclin dependent kinase (CDK)2 and 4 were down-regulated significantly, whereas the pressionlevels of cell cycle dependent kinase inhibitors (CDKI) including p16, p19, p27and p53 were up-regulated markedly in skin tissues from PTHrP KI mice. These results suggest that skin senesence occurred in PTHrP KI mice is associated with inhibiting dermatic cell proliferation and enhancing dermatic cell apoptosis through the down-regulation of cyclin D1 and E and CDK2 and 4 and the up-regulation of CDKI including p16, p19, p27and p53.The density of skin vessels was reduced in the PTHrP KI mice compared to WT littermates demonstrated by imaging with steromicroscopy in fresh skin samples and the immunohistochemical staining for platelet endothelial cell adhesion molecule-1 (CD31). The gene expression levels of CD31,vascular endothelial growth factor (VEGF), angiopoietin-1(Ang-1), angiopoietin-2(Ang-2) and endothelial cell receptor tyrosine kinases(Tie-2) were down-regulated significantly at the postnatal day 1, 7 and 14 in skin tissues from PTHrP KI mice demonstrated by the real-time RT-PCR. Consistent with gene expression, the protein expression levels of CD31, VEGF, Ang-1 and Ang-2 were also down-regulated in skin tissues from PTHrP KI mice demonstrated by Western blot. These results suggest that skin senesence occurred in PTHrP KI mice is also associated with the impairment of angiogenesis in the skin. According to our previous studys that the expression level of Bmi-1 and the nuclear translocation were reduced in mouse embryonic fibroblasts (MEFs) from PTHrP KI mice, we proposed whether Bmi-1 was as a down-stream target of PTHrP mediated skin senesence caused by PTHrP KI. To test our hypothesis, the interaction of PTHrP with Bmi-1 was assessed by co-immunoprecipitation and the dermatic phenotypes were compared between the wild-type and Bmi-1 knockout (KO) mice at 2 and 4 weeks of age using morphological, cellular and molecular approaches. The total collagen positive area was reduced significantly at 2-week-old Bmi-1 KO mice. The skin was thinner with hyperkeratosis of the epidermis, collagen and subcutaneous fat deposition in skin matrix were reduced in 4-week-old Bmi-1 KO mice compared to their WT littermates. The proliferation of dermatic cells was decreased demonstrated by immunohistochemical staining for PCNA in 2- and 4-week-old Bmi1 KO mice. The expression levels of cell cycle dependent kinase inhibitors including p16,p19 and p27 were up-regulated markedly in skin tissues from Bmi-1 KO mice demonstrated by Western blot. These findings indicates that Bmi-1 may be as a down-stream target of PTHrP mediated skin senesence caused by PTHrP KI by inhibiting the proliferation of dermatic cells and stimulating the apoptosis of dermatic cells through the up-regulation of CDKI including p16, p19, p27.This study demonstrated that PTHrP NLS and carboxyl terminus plays an important role in preventing skin aging by stimulating the proliferation of dermatic cells, inhibiting the apoptosis of dermatic cells and enhancing angiogenesis of the skin mediated by Bmi-1. This study provides an experimental and theoretical evidence that PTHrP NLS and carboxyl terminus could be used as a therapeutics to prevent skin aging.