Severe growth retardation and early lethality in mice lacking the nuclear localization sequence and C-terminus of PTH-related protein - PubMed (original) (raw)

Dengshun Miao et al. Proc Natl Acad Sci U S A. 2008.

Erratum in

Abstract

Parathyroid hormone (PTH) plays a central role in the regulation of serum calcium and phosphorus homeostasis, while parathyroid hormone-related protein (PTHrP) has important developmental roles. Both peptides signal through the same G protein-coupled receptor, the PTH/PTHrP or PTH type 1 receptor (PTH1R). PTHrP, normally a secreted protein, also contains a nuclear localization signal (NLS) that in vitro imparts functionality to the protein at the level of the nucleus. We investigated this functionality in vivo by introducing a premature termination codon in Pthrp in ES cells and generating mice that express PTHrP (1-84), a truncated form of the protein that is missing the NLS and the C-terminal region of the protein but can still signal through its cell surface receptor. Mice homozygous for the knock-in mutation (Pthrp KI) displayed retarded growth, early senescence, and malnutrition leading postnatally to their rapid demise. Decreased cellular proliferative capacity and increased apoptosis in multiple tissues including bone and bone marrow cells were associated with altered expression and subcellular distribution of the senescence-associated tumor suppressor proteins p16(INK4a) and p21 and the oncogenes Cyclin D, pRb, and Bmi-1. These findings provide in vivo experimental proof that substantiates the biologic relevance of the NLS and C-terminal portion of PTHrP, a polypeptide ligand that signals mainly via a cell surface G protein-coupled receptor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Pthrp KI mice exhibit growth retardation and premature aging. (A) Growth curves of wild-type (WT) and Pthrp KI mice. (B) Whole mount skeletons of newborn WT and Pthrp KI mice stained with alcian blue (for cartilage) and alizarin red (for calcified tissue). (C) Surviving KI mice at 14 days of age show osteopenia and severe kyphosis (arrow). (D) KI mice at day 14 show loss of body mass, muscle atrophy, and a profound decrease in adipose tissue (Ad). (E) Representative micrographs of brains showing diffuse swelling of the tissue with complete obliteration of the sulci in the KI mice. (F) Representative micrographs of skin from Pthrp KI mice showing thinner skin (green line) with hyperkeratosis of the epidermis (blue line) (magnification, ×100). (G) Representative micrographs of common carotid arteries (magnification, ×400). (H) Representative micrographs of renal tissue sections showing endogenous β-galactosidase activity in renal cortical tubules from Pthrp KI mice, but not from WT mice (magnification, ×400). (I) Contact radiographs (Left) and microCT 3D reconstruction (Right) of the mandibles from WT and Pthrp KI mice (Left and Right specimen in each panel, respectively). (J) Western blot analysis of skeletal muscle extracts for LC3-I and II. ß-Tubulin was used as loading control.

Fig. 2.

Fig. 2.

Skeletal growth retardation caused by impaired growth plate chondrocyte proliferation. (A) Representative micrographs of H&E stained sections of the proximal ends of tibiae from E18.5 wild-type (WT) and Pthrp KI mice (magnification, ×100). Blue lines represent the respective proliferating zones. (B) The length measurements of the proliferating zone. (C) Representative contact radiographs of the femurs of WT and Pthrp KI mice at 2 weeks of age. (D) Femoral length measurements. (E) BMD measurements. (F) Representative frontal views of microCT 3D reconstruction of the proximal end of tibiae. (G) Epiphyseal volume of the proximal ends of tibiae. (H) Width of the cartilaginous growth plate. (I) Undecalcified sections of tibiae stained by the von Kossa procedure (magnification, ×200). (J) Mineralized area, percentage of growth plate. (K) Paraffin-embedded sections of tibiae from WT and KI mice stained immunohistochemically for PCNA (arrowheads) (magnification, ×400). (L) Number of PCNA-positive chondrocytes as a percentage of total chondrocytes, as determined by image analysis. (M) Immunostaining for type X collagen (arrowheads) (magnification, ×100). (N) Width of type X collagen-positive hypertrophic zone of growth plates. Data shown represent mean ± SE of five animals per group. **, P < 0.01; ***, P < 0.001 in the KI mice relative to the wild-type mice.

Fig. 3.

Fig. 3.

Severe premature osteoporosis resulted from defects of osteoblastic bone formation. (A) Representative micrographs of hematoxylin and eosin (H&E) stained sections of the metaphyseal region of tibiae from E18.5 wild-type (WT) and Pthrp KI mice (magnification, ×400). (B) Osteoblast number/tissue area (N.Ob/T.Ar), (C) Osteoblast surface/bone surface ratio (Ob.S/BS). (D) Representative longitudinal (top) and cross sections (bottom) of 3D reconstruction of proximal end of tibiae at 2 weeks of age. Quantitative histomorphometry for (E) bone volume/total volume (BV/TV), (F) cortical bone volume (Ct.V.), (G) trabecular number (Tb.N), (H) trabecular thickness (Tb.Th), and (I) trabecular separation (Tb.Sp). (J) Representative micrographs of tibial sections from the WT and KI mice stained histochemically for TRAP (magnification, ×200), (K) Osteoclast number/tissue area (N.Oc/T.Ar), (L) Representative micrographs of tibial sections from the WT and KI mice stained immunohistochemically for Pth1r (magnification, ×400), (M) Pth1r positive area as percent of tissue. (N) Representative micrographs of tibial sections from the WT and KI mice stained with H&E (magnification, ×400). (O) Osteoblast number/tissue area (N.Ob/T.Ar). (P) Western blot of long bone extracts for the expression of Cbfa1 and Pth1r. ß-tubulin was used as loading control for Western blots. Data shown represent mean ± SE of five animals per group. ***, P < 0.001 in the KI mice relative to wild-type littermates.

Fig. 4.

Fig. 4.

The Pthrp KI phenotype is associated with inhibition of cell proliferation and stimulation of cell apoptosis. (A) Representative micrographs of sections from the subventricular zone (SVZ) and the hippocampus (HP) of brains from E18.5 WT and KI mice immunostained for PCNA (brown color indicated by arrowheads; magnification, ×400). (B) PCNA positive cell numbers in SVZ and HP (number/per field). (C) Bone marrow cells from 14-day-old Pthrp KI mice show decreased incorporation of BrdU (red) compared to those from WT mice. (D) Quantitative assessment of BrdU incorporation using flow cytometry (blue profile for negative control, red profile for BrdU positive cells). (E) Representative micrographs of thymus sections from WT and KI mice stained for apoptotic cells using the TUNEL technique (red, magnification, ×1000). (F) Flow cytometry analysis of apoptotic thymocytes (Annexin-V positive (+)/PI negative (−) cells; blue profile for PI positive cells, red profile for Annexin-V positive cells). (G) Representative spleen sections from WT and KI mice stained for apoptotic cells using the TUNEL technique (red, magnification, ×1000). (H) Flow cytometry analysis of apoptotic spenocytes (blue profile for PI positive cells, red profile for Annexin-V positive cells). (I) The percentage of apoptotic cells in thymus and spleen as determined by TUNEL assay were quantified by image analysis and are presented as mean ± SE of triplicate determinations. (J) The percentage of Annexin-V positive (+)/PI negative (−) cells in thymus and spleen was quantified from flow cytometry analysis. Data shown represent mean ± SE from five animals per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for the Pthrp KI samples relative to those from wild-type littermates. (K) Western blot analysis of thymus and spleen extracts for Bax and Bcl-2. ß-Tubulin was used as loading control.

Fig. 5.

Fig. 5.

Altered expression and subcellular distribution of senescence-associated tumor suppressor proteins and oncogenes. (A) Western blot analysis of long bone and MEF extracts for p16INK4a, p21, and Bmi-1. ß-Tubulin was used as loading control. (B) MEFs cultured for 14 days in DMEM with 10% FCS were stained immunocytochemically for p16INK4a, p21, and Bmi-1 (red; magnification, ×1000). (C) Western blot analysis of long bone extracts for Cyclin D, Cdk4, Cdk6, and phosphorylated Rb (pRb). ß-Tubulin was used as loading control. (E) Proposed regulation of proliferation and senescence in cells that expresses either PTHrP (1–139) (Left) or PTHrP (1–84) which lacks the NTS and the C terminus (CT) (Right). In the absence of NTS and C terminus PTHrP, Bmi-1 remains inactive in the cytoplasm, leading to increased p16INK4a (as well as p21) levels, G1 cell-cycle arrest and senescence.

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