Severe Growth Retardation and Early Lethality in Mice Lacking the Nuclear Localization Sequence and C-Terminus of PTH-Related Protein

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

Dengshun Miaoa,b, Hanyi Suc, Bin Hec, Jianjun Gaob, Qingwen Xiab, Min Zhua, Zhen Gua, David Goltzmanb, and Andrew C. Karaplisc,1

aDepartment of Anatomy, Histology and Embryology, The Research Center for Bone and Stem Cells, Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of China; and bCalcium Research Laboratory and Department of Medicine, McGill University Health Centre, McGill University, Montreal, Canada H3A 1A1; cDivision of Endocrinology, Department of Medicine and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, Canada H3T 1E2

Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved October 29, 2008 (received for review June 12, 2008) Parathyroid hormone (PTH) plays a central role in the regulation of cell-cycle dependent (22) in that PTHrP is targeted to the nucleolus serum calcium and phosphorus homeostasis, while parathyroid hor- of cells in G1. mone-related protein (PTHrP) has important developmental roles. In the present study, we examined the potential biological Both peptides signal through the same G protein-coupled receptor, relevance of the nuclear localization of PTHrP in vivo by inserting the PTH/PTHrP or PTH type 1 receptor (PTH1R). PTHrP, normally a a premature termination codon (TGA) in the murine Pthrp, and secreted protein, also contains a nuclear localization signal (NLS) that generating a ‘‘knock-in’’ (KI) mouse expressing PTHrP (1–84), a in vitro imparts functionality to the protein at the level of the nucleus. form of the protein that lacks the NLS and C-terminal region. Using We investigated this functionality in vivo by introducing a premature this approach, Pthrp is expected to be expressed in relevant cells and termination codon in Pthrp in ES cells and generating mice that at physiologic levels. In addition, the truncated PTHrP protein express PTHrP (1–84), a truncated form of the protein that is missing should be processed and secreted freely within tissues to bring about the NLS and the C-terminal region of the protein but can still signal the appropriate paracrine/autocrine effects by interacting with through its cell surface receptor. Mice homozygous for the knock-in PTH1R, which localizes at the plasma membrane but would be mutation (Pthrp KI) displayed retarded growth, early senescence, and devoid of any potential nuclear actions. malnutrition leading postnatally to their rapid demise. Decreased Results cellular proliferative capacity and increased apoptosis in multiple tissues including bone and bone marrow cells were associated with Generation of a ‘‘Knock-In’’ (KI) Mouse Expressing PTHrP (1–84). We altered expression and subcellular distribution of the senescence- first targeted the Pthrp locus in ES cells by introducing a premature termination codon at amino acid position 85 of the encoded mature associated tumor suppressor proteins p16INK4a and p21 and the protein [see supporting information (SI) Fig. S1 A–D]. Appropri- oncogenes Cyclin D, pRb, and Bmi-1. These ﬁndings provide in vivo CELL BIOLOGY ately targeted ES cells from two clones containing the mutated experimental proof that substantiates the biologic relevance of the Pthrp gene were microinjected into 3.5-day C57BL/6 blastocysts and NLS and C-terminal portion of PTHrP, a polypeptide ligand that signals then transferred into uteri of 2.5-day post coitus pseudopregnant mainly via a cell surface G protein-coupled receptor. CD1 mice to generate chimeric animals. Extensively chimeric male mice were crossed to C57BL/6 female mice and heterozygous ͉ ͉ ͉ ͉ ageing nucleus osteoporosis PTHrP senescence offspring were identified by PCR of tail genomic DNA (Fig. S1E). They, in turn, were bred to obtain animals homozygous for the ntranuclear transport of numerous polypeptide ligands has been mutated Pthrp allele (Pthrp KI mice). Absence of PTHrP (1–84) Ireported, yet the function of these ligands at the level of the truncated mutant from the nucleus was confirmed in mouse nucleus and its biological relevance in the in vivo setting remain in embryonic fibroblasts (MEFs) derived from these mice and appro- question (1–3). The parathyroid hormone-related protein (Pthrp) priate expression levels were verified (Fig. S2 A-D). gene encodes a nuclear localization signal (NLS) within the 87 to 107 region of the mature protein product and contains at least two Pthrp KI Mice Exhibit Growth Retardation and Premature Aging. The translational initiation sites, one that generates a conventional phenotype of Pthrp KI mice was distinct from that of homozygous signal peptide and one that disrupts it (4, 5). These features allow Pthrp-null mice (11, 12, 23). At birth, Pthrp KI mice were similar in PTHrP either to be secreted in a paracrine/autocrine fashion or to weight to wild-type littermates. However, by three days postpartum be retained within the cytosol and to be translocated to the nucleus they failed to grow relative to their wild-type littermates (Fig. 1A) (4, 6, 7). Alternative potential mechanisms of PTHrP entry into the and died by two to three weeks of age. Serum levels for calcium, cytosol have also been described (8–10). phosphorus, and PTH were normal at two weeks of age (Fig. S2 The similarity of the N terminus of PTHrP to that of parathyroid E-G). The Pthrp KI mice were slightly smaller than wild-type hormone (PTH), the major hormone regulator of calcium and littermates without markedly shorter limbs at birth, a distinguishing phosphorus homeostasis, enables PTHrP to share the signaling feature of the homozygous null mice (Fig. 1B). At 2 weeks, the Pthrp properties of PTH by interacting with the common PTH/PTHrP or

PTH type 1 receptor (PTH1R), a member of the G protein coupled Author contributions: D.G. and A.C.K. designed research; D.M., H.S., B.H., J.G., Q.X., M.Z., receptor family B. Previous studies have clearly documented the and Z.G. performed research; D.M., D.G., and A.C.K. analyzed data; and D.M., D.G., and indispensable biological importance of PTHrP/PTH1R signaling in A.C.K. wrote the paper. skeletal (11–15) and mammary gland (16, 17) development. Addi- The authors declare no conﬂict of interest. tional endocrine and paracrine functions have been ascribed to the This article is a PNAS Direct Submission. mid-region and C-terminal region (107 to 139) of the molecule (18, 1To whom correspondence should be addressed. E-mail: [email protected]. 19). In vitro studies have indicated that PTHrP displays other This article contains supporting information online at www.pnas.org/cgi/content/full/ functions largely relating to an intracrine signaling role in the 0805690105/DCSupplemental. nucleus/nucleolus (4, 20, 21) and that its subcellular distribution is © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805690105 PNAS ͉ December 23, 2008 ͉ vol. 105 ͉ no. 51 ͉ 20309–20314 Downloaded by guest on September 26, 2021 Fig. 1. Pthrp KI mice exhibit A B WT KI C WT KI D WT KI growth retardation and premature Ad 5 aging. (A) Growth curves of wild- WT KI *** type (WT) and Pthrp KI mice. (B) Whole mount skeletons of new- 4 Ad born WT and Pthrp KI mice stained with alcian blue (for cartilage) and 3 alizarin red (for calciﬁed tissue). (C) * Surviving KI mice at 14 days of age Body weight (g) 2 show osteopenia and severe kyphosis (arrow). (D) KI mice at day 14 1 show loss of body mass, muscle at- 1237 rophy, and a profound decrease in Age (days) adipose tissue (Ad). (E) Representa- WT KI tive micrographs of brains showing E F I diffuse swelling of the tissue with I complete obliteration of the sulci in WT KI Skin the KI mice. (F) Representative micrographs of skin from Pthrp KI mice showing thinner skin (green line) with hyperkeratosis of the epider- G mis (blue line) (magniﬁcation, J

ϫ100). (G) Representative micro- WT-fed WT-starved KI-fed Carotid artery graphs of common carotid arteries LC3-I (magniﬁcation, ϫ400). (H) Repre- LC3-II sentative micrographs of renal tis- H sue sections showing endogenous β ␤-galactosidase activity in renal cor- -Tubulin

tical tubules from Pthrp KI mice, but galactosidase β− not from WT mice (magniﬁcation, ϫ400). (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.

KI mice exhibited marked phenotypic changes indicative of pre- rable to the degrees of autophagy observed in other progeroid mature aging including an unstable gait, cachexia, osteopenia with mouse models (32). kyphosis (Fig. 1C), and a profound decrease in fat deposition (Fig. 1D). The brains of these animals were both smaller and edematous Skeletal Growth Retardation Caused by Impairment in Endochondral (Fig. 1E). The skin was thin with hyperkeratosis of the epidermis Bone Formation. In E18.5 Pthrp KI mice, growth plates were reduced (Fig. 1F) and large blood vessels were often atrophic and therefore in size with narrower proliferative zones (Fig. 2 A and B), with no potentially more subject to rupture (Fig. 1G). Nevertheless, it was evidence of the markedly disrupted architecture in the hypertrophic difficult to identify a single cause of death in our animals and indeed zone characteristic of the growth plates of Pthrp-null mice (12). In premature senescence often leads to death from multiorgan failure 2-week-old Pthrp KI mice, long bones were markedly shorter (Fig. (24). The senescence biomarker ␤-galactosidase (25) was present in 2 C and D) and osteoporotic, as determined by decreased bone tissues such as kidney (Fig. 1H) and lung (data not shown), mineral density (Fig. 2E), while epiphyseal volumes were reduced, suggesting that these tissues undergo early onset senescence. This resulting in small growth plates, as verified by microCT imaging and early senescence phenotype contrasts strikingly to the phenotypes histology (Fig. 2 F-H). Cartilaginous matrix mineralization, how- of the Pthrp-null (11, 12) and those of the Pth1r-null mice (13). ever, was appropriate (Fig. 2 I and J). The proliferation of chondrocytes, as determined by immunostaining for proliferating cell Influence of Nutrition on the Phenotype of the Pthrp KI Mice. We next nuclear antigen (PCNA), was profoundly diminished in the KI mice assessed the potential influence of nutrition on the phenotype of the (Fig. 2 K and L). Staining also decreased for type X collagen, a Pthrp KI mice. Stomach milk weights (Fig. S3A) were similar during marker of terminal chondrocyte differentiation (Fig. 2 M and N). the first two days but lower with time, although milk intake relative However, the pattern of cellular differentiation within the growth to body weight was not significantly reduced after day 1 (Fig. S3B). plate was normal. This therefore reflected a reduction in differen- Blood glucose levels were reduced (Fig. S3C) in keeping with tiated chondrocytes, which was likely secondary to their decreased previous observations in other early senescent models (26–28). proliferative capacity. This phenotype, although resulting in skeletal Interestingly, in contrast to the Pthrp-null mice (29), tooth eruption growth retardation, stood in striking contrast to the chondrodys- occurred in the Pthrp KI mice (Fig. 1I). plastic Pthrp-null long bones, where the disorganized hypertrophic We then sought to compare the presence of autophagy in zone is secondary to premature and inappropriate differentiation of 4-day-old wild-type fed mice, 4-day-old wild-type mice that had chondrocytes (12). been starved for 24 h, and 4-day-old Pthrp KI fed mice. Immuno- blots of microtuble-associated protein light chain 3 (LC3-I), an Premature Osteoporosis Results from Defective Osteoblastic Bone For- autophagosome marker protein (30, 31), were performed in skel- mation. The trabecular bone of E18.5 Pthrp KI mice showed reduced etal muscle, a major site of the manifestation of malnutrition. LC3-I osteoblasts (Fig. 3 A-C). In 2 week old mice, the longitudinal and was only modestly converted to membrane bound (lipidated) LC3 cross sectional views from 3-D microCT reconstructions of long (LC3-II) in fed wild-type mice but was markedly converted in the bones further confirmed the osteoporotic phenotype of the Pthrp starved wild-type mice (Fig. 1J). In contrast, levels of LC3-II KI mice (Fig. 3D). Trabecular and cortical bone volumes, and relative to LC3-I in the Pthrp KI mice were greater than in the fed trabecular number and thickness were decreased, whereas trabec- wild-type, but substantially lower than in the starved mice (Fig. S3 ular spacing was increased (Fig. 3 E-I). Tartrate resistant acid D and E). Although undernourishment may have contributed in phosphatase (TRAP) activity and the number of osteoclasts were part to the phenotype, these levels in Pthrp KI mice were compa- decreased in mutant bones (Fig. 3 J and K). The immunopositive

20310 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805690105 Miao et al. Downloaded by guest on September 26, 2021 WT KI B A WT KI B 800 WT C KI

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10 bone formation. (A) Representative micrographs of hematoxylin and eosin CELL BIOLOGY 8 6 (H&E) stained sections of the metaphyseal region of tibiae from E18.5 wild- 4 ϫ 2 type (WT) and Pthrp KI mice (magniﬁcation, 400). (B) Osteoblast number/ Mineralized area (% of growth plate) 0 tissue area (N.Ob/T.Ar), (C) Osteoblast surface/bone surface ratio (Ob.S/BS). (D) K Representative longitudinal (top) and cross sections (bottom) of 3D recon- L 25 struction of proximal end of tibiae at 2 weeks of age. Quantitative histomor- 20 phometry for (E) bone volume/total volume (BV/TV), (F) cortical bone volume 15 (Ct.V.), (G) trabecular number (Tb.N), (H) trabecular thickness (Tb.Th), and (I) 10 trabecular separation (Tb.Sp). (J) Representative micrographs of tibial sections

(% of total) *** 5 from the WT and KI mice stained histochemically for TRAP (magnification, 0 ϫ200), (K) Osteoclast number/tissue area (N.Oc/T.Ar), (L) Representative mi- M +ve chondrocytes PCNA crographs of tibial sections from the WT and KI mice stained immunohisto- N 300 chemically for Pth1r (magnification, ϫ400), (M) Pth1r positive area as percent 250 of tissue. (N) Representative micrographs of tibial sections from the WT and KI 200 mice stained with H&E (magnification, ϫ400). (O) Osteoblast number/tissue 150 *** 100 area (N.Ob/T.Ar). (P) Western blot of long bone extracts for the expression of 50 Cbfa1 and Pth1r. ß-tubulin was used as loading control for Western blots. Data Width of HZ ( µ m) 0 shown represent mean Ϯ SE of five animals per group. ***, P Ͻ 0.001 in the KI mice relative to wild-type littermates. 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 (magnifi- area for osteoblastic PTH1R was also dramatically decreased in ϫ cation, 100). Blue lines represent the respective proliferating zones. (B) The long bone sections from Pthrp KI mice (Fig. 3 L and M) and length measurements of the proliferating zone. (C) Representative contact ra- associated with a marked reduction in osteoblast number (Fig. 3 N diographs 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 and O) and protein expression of Cbfa1 and Pth1r (Fig. 3P). We of microCT 3D reconstruction of the proximal end of tibiae. (G) Epiphyseal volume therefore concluded that decreased osteoblastic bone formation of the proximal ends of tibiae. (H) Width of the cartilaginous growth plate. (I) was the major cause of the osteoporotic phenotype in the Pthrp KI Undecalcified sections of tibiae stained by the von Kossa procedure (magnifica- mice. This bone phenotype was not secondary to altered external tion, ϫ200). (J) Mineralized area, percentage of growth plate. (K) Paraffin- humoral or nutritional factors, as determined by bone transplan- embedded sections of tibiae from WT and KI mice stained immunohistochemi- tation experiments (Fig. S4 A and B). cally for PCNA (arrowheads) (magnification, ϫ400). (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, Reduced Cell Proliferation and Increased Cellular Apoptosis in Pthrp KI ϫ100). (N) Width of type X collagen-positive hypertrophic zone of growth plates. Tissues. In addition to the reduced numbers of proliferative chon- Data shown represent mean Ϯ SE of five animals per group. **, P Ͻ 0.01; ***, P Ͻ drocytes and osteoblasts in situ, in utero, we observed a profound 0.001 in the KI mice relative to the wild-type mice. decrease in PCNA positive stem/progenitor cells in the subven-

Miao et al. PNAS ͉ December 23, 2008 ͉ vol. 105 ͉ no. 51 ͉ 20311 Downloaded by guest on September 26, 2021 WT KI A B 800 700 WT KI 600 500 SVZ 400 *** 300 200 *** PCNA positive cell number PCNA in SVZ and HP (#/per field) (#/per in and HP SVZ 100 0 SVZ HP HP D WT KI

C Fig. 4. The Pthrp KI phenotype is associated with

BM inhibition of cell proliferation and stimulation of cell apoptosis. (A) Representative micrographs of sections C itive from the subventricular zone (SVZ) and the hippocampus (HP) of brains from E18.5 WT and KI mice immu-

BrdU Pos nostained for PCNA (brown color indicated by arrow- ϫ BM (BrdU) heads; magniﬁcation, 400). (B) PCNA positive cell numbers in SVZ and HP (number/per ﬁeld). (C) Bone F marrow cells from 14-day-old Pthrp KI mice show de- -

I creased incorporation of BrdU (red) compared to

;P those from WT mice. (D) Quantitative assessment of E + BrdU incorporation using flow cytometry (blue profile nV for negative control, red profile for BrdU positive

thymocytes cells). (E) Representative micrographs of thymus sec- Annexi

Thymus tions from WT and KI mice stained for apoptotic cells using the TUNEL technique (red, magnification, ϫ1000). (F) Flow cytometry analysis of apoptotic thy- H mocytes (Annexin-V positive (ϩ)/PI negative (Ϫ) cells; G - blue profile for PI positive cells, red profile for An- ;PI

+ nexin-V positive cells). (G) Representative spleen sections from WT and KI mice stained for apoptotic cells using the TUNEL technique (red, magnification, Spleen splenocytes ϫ1000). (H) Flow cytometry analysis of apoptotic spe- Annexin V nocytes (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 Thymus Spleen TUNEL assay were quantified by image analysis and 8 ) 16 * K IJ*** WT KI WT KI Ϯ 7 (% 14 are presented as mean SE of triplicate determina- ls 6 l 12 ** tions. (J) The percentage of Annexin-V positive (ϩ)/PI *** e Bax c s(%)

- Ϫ ll 5 I 10 negative ( ) cells in thymus and spleen was quantified e 4 P 8 from flow cytometry analysis. Data shown represent cc +; ti V o 3 6 Bcl-2 mean Ϯ SE from five animals per group. *, P Ͻ 0.05; **,

xin 4 P Ͻ 0.01; , P Ͻ 0.001 for the Pthrp KI samples 2 e *** Apopt

1 nn 2 relative to those from wild-type littermates. (K) West- A 0 0 β-Tubulin ern blot analysis of thymus and spleen extracts for Bax Thymus Spleen Thymus Spleen and Bcl-2. ß-Tubulin was used as loading control.

tricular zone and the hippocampus in brains from E18.5 Pthrp KI (Pc-G/trx-G) proteins (33, 34). Indeed, expression of p16INK4a and mice (Fig. 4 A and B). In 14-day-old mutant mice, bone marrow cells p21 was significantly increased, while that of Bmi-1 was reduced in showed significantly lower incorporation of BrdU compared to tissues (including bone, thymus, and spleen) and in MEFs derived their wild-type littermates (40.0% vs. 66.3%) (Fig. 4 C and D). from Pthrp KI mice compared to wild-type counterparts even when Moreover, TUNEL-positive cells and annexin Vϩ/propidium io- mutant MEFs were cultured under conditions of full nutritional dide-negative (PIϪ) cells were increased in the thymus and spleen supplementation (Fig. 5A). Furthermore, nuclear localization of of Pthrp KI mice compared to wild-type littermates (Fig. 4 E-J). p16INK4a and p21 was increased in MEFs from KI mice (Fig. 5B). Consistent with increased cellular apoptosis in Pthrp KI mice, In contrast, Bmi-1 was detected in the nuclei of MEFs from expression of the proapoptotic protein Bax was dramatically in- wild-type but not in nuclei of MEFs derived from Pthrp KI mice. In creased, whereas that of the anti-apoptotic protein Bcl-2 was addition, levels of Cyclin D, Cdk4, and Cdk6 and the phosphory- decreased (Fig. 4K), suggesting that the observed growth retarda- lated form of the retinoblastoma gene product (pRb), all of which tion and in vivo senescence phenotypes were associated with are essential for cell proliferation and have been found to be altered inhibition of cell proliferation and stimulation of cellular apoptosis. in senescent states (35) were all reduced (Fig. 5C).

Altered Senescence-Associated Tumor Suppressor Genes and Oncogene Discussion Expression in Pthrp KI Mice. In view of the fact that senescence is the Our findings indicate that in vivo deletion of the NLS and C- final phenotypic state of decreased cell proliferation often mediated terminal region of PTHrP leads to growth retardation and early by increased expression and activation of tumor suppressor genes, senescence by altering expression patterns and subcellular distri- we examined the expression of p16 INK4a and p21 as well as that of bution of proliferative and senescence-related genes in multiple the oncogene Bmi-1, a member of the Polycomb/trixthorax group tissues. Although malnutrition manifested by autophagy may have

20312 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805690105 Miao et al. Downloaded by guest on September 26, 2021 WT KI and to also stimulate (44) or inhibit (45) apoptosis. The biological A Bone MEFs B WT KI WT KI INK4a actions of PTH1R apparently involved in longevity (as well as in p16 INK4a p16 growth plate and tooth eruption) most likely reside in local actions of the N terminus of PTHrP rather than in circulating PTH, in view of the p21 p21 fact that Pth null mice do not exhibit a reduced life span (23) and PTH levels were normal in our model. These distinct actions of the N- Bmi-1 terminal domains of PTH and PTHrP may reflect conformational Bmi-1 β-Tubulin selectivity for the PTH1R, as recently described (46).

C D Modulation of Cell Cycle and Apoptosis by PTHrP. In vitro studies PTHrP (1-139) PTHrP (1-84) investigating the cellular mechanisms responsible for the mitogenic checkpoint release by nuclear PTHrP have shown that in vascular Plasma WT KI membrane PTHR1 smooth muscle cells the NLS, together with the C-terminal region Cyclin D Bmi-1 of PTHrP, is translocated to the nucleus of cells in G1 (22). The Cytoplasm Cdk4 nuclear presence of PTHrP appears to trigger pRb phosphorylation and release of G1/S arrest and thereby cell cycle progression (21). Cdk6 Nucleus PTHrP (1-139) or NTS-CT Bmi -1 INK4a In this setting, cyclin E/Cdk-2 kinase activity is markedly increased Bmi- INK4a Senescence p16 Senescence pRb Bmi-11 p16 by PTHrP as a result of marked PTHrP-induced proteasomal p21 kip1 CyclinD/Cdk4/6 CyclinD/Cdk4/6 degradation of p27 (47). In contrast, we show here that in Pthrp β-Tubulin pRb KI cells, one consequence of the absence of nuclear PTHrP action pRb in vivo is increased p21 levels, which in turn would lead to inhibition Cell-cycle progression & self-renewal G cell-cycle arrest & senescence 1 of cyclin E/Cdk2 and cyclin D1/Cdk4/Cdk6 activities (shown to be reduced in our studies) and to cell-cycle arrest in G1 phase. Fig. 5. Altered expression and subcellular distribution of senescence- PTHrP has also been shown to bind to RNA (48). In view of associated tumor suppressor proteins and oncogenes. (A) Western blot anal- evidence that some rDNA transcription occurs largely at the ysis of long bone and MEF extracts for p16INK4a, p21, and Bmi-1. ß-Tubulin was border between the fibrillar center and the dense fibrillar used as loading control. (B) MEFs cultured for 14 days in DMEM with 10% FCS component of the nucleolus (49) where PTHrP localizes in vivo were stained immunocytochemically for p16INK4a, p21, and Bmi-1 (red; mag- ϫ (4), it is conceivable that it partakes in regulating a variety of niﬁcation, 1000). (C) Western blot analysis of long bone extracts for Cyclin D, nucleolar functions required to support cell growth and division, Cdk4, Cdk6, and phosphorylated Rb (pRb). ß-Tubulin was used as loading control. (E) Proposed regulation of proliferation and senescence in cells that and coordination of cellular stress responses (50). expresses either PTHrP (1–139) (Left) or PTHrP (1–84) which lacks the NTS and Bmi-1 is required for the maintenance of adult stem cells the C terminus (CT) (Right). In the absence of NTS and C terminus PTHrP, Bmi-1 because it promotes cell proliferation and suppresses genes that remains inactive in the cytoplasm, leading to increased p16INK4a (as well as p21) induce cellular senescence (33) and cell death (34). Its action is levels, G1 cell-cycle arrest and senescence. due in part to its ability to suppress expression of proteins that inhibit cell cycle progression such as p16INK4a (33). Expression of p16INK4a rises markedly with aging in many tissues (51), inhibits CELL BIOLOGY contributed in part to the observed phenotype, the Pthrp KI mouse cyclin D-dependent kinases, and prevents phosphorylation of ␤ is a progeroid mouse model as deposition of -galactosidase, Rb, thus limiting proliferation and self-renewal (50). This re- regarded as a senescence marker even in the presence of autophagy duces the reservoirs of self-renewing tissue stem cells required (36), was detected in its tissues. to regenerate lost or damaged cells with aging. We have shown here that p16INK4a is highly expressed in tissues from Pthrp KI Interactions of the PTHrP Domains. Previous in vivo studies have mice and that phosphorylated Rb is reduced suggesting that reported that Pthrp null mice (11, 12) as well as Pth1r null mice (13), Bmi-1 function is impaired. demonstrate dysplastic long bone formation and early lethality (i.e., Nuclear translocation of Bmi-1 is necessary for its function in utero or within one to two days following birth). Targeted (52, 53), and Bmi-1 that retains its cytoplasmic localization is overexpression of PTHrP in chondrocytes of Pthrp null mice (37) inactive (52). In our studies, Bmi-1 localized to the nucleus in largely eliminated the growth plate pathology as did targeted wild-type cells, but not in cells derived from Pthrp KI mice, most overexpression of PTH1R in chondrocytes of Pthrp (38) and Pth1r likely resulting in failure to suppress p16INK4a expression in Pthrp null mice (39). Both models displayed reduced survival despite KI MEFs and tissues. It is probable that the nucleocytoplasmic improvement in long bones, however, indicating that the defect in shuttling ability of PTHrP (54, 55), which is determined by its viability does not result from the skeletal abnormalities, but de- nuclear localizing domain, is involved in the nuclear shuttling of tailed descriptions of extra-skeletal pathology have not been avail- proteins such as Bmi-1 and disruption of this mechanism results able. The presence of a markedly distinct phenotype in the long in inactive Bmi-1 (Fig. 5D). Further studies will be required to bones and teeth of our Pthrp KI mice supports the unique action of delineate the importance of these actions in the regulation of the nuclear localizing domain of PTHrP in skeletal pathology. The cellular proliferation and senescence. early lethality in Pth1r null mice (13) as well as in our mice lacking the nuclear localizing/C-terminal domains, may indicate that Biological Implications. We propose that the Pthrp KI mouse is the unique mechanisms may exist for PTH1R and for the nuclear first laboratory animal model with a distinct phenotype arising from localizing domain to promote optimal survival, and the absence of the inability of a peptide hormone to act within the nucleus while both regions, in the Pthrp null mice may co-operatively contribute maintaining its role as a secreted ligand. Undoubtedly, these studies to the more rapid death of these animals than of the mice lacking do not shed light on whether the observed phenotypic alterations the nuclear localizing/C-terminal domains. In this respect, the latter are exclusively a consequence of loss of C-terminal PTHrP action PTHrP domains have been implicated in enhancement of prolif- at the level of the nucleus or elsewhere in the cell. Our conclusions, eration (6, 20, 21), a function reported to require both an intact NLS however, are corroborated by in vitro observations showing that and an intact C-terminal region (20), and in inhibition of apoptosis activation of cell proliferation requires both an intact NLS and an (4, 6, 40, 41) in several cell systems in vitro. In contrast, the amino intact C terminus and that deletion of the NLS prevents nuclear (N) terminal domain has been reported to either stimulate (42) or entry and slows proliferation (20). The demonstration here of the inhibit (6, 43) proliferation depending on the in vitro system used, functionality of the NLS and C terminus of PTHrP at the tissue and

Miao et al. PNAS ͉ December 23, 2008 ͉ vol. 105 ͉ no. 51 ͉ 20313 Downloaded by guest on September 26, 2021 organismal level now adds further credence to the biologic rele- for 30 sec, and 72 °C for 45 sec (35 cycles), followed by an extension at 72 °C for vance of nuclear transport and function of polypeptide ligands. 10 min. PCR products were digested with BstEII at 60 °C for 2 h and analyzed by agarose gel electrophoresis. The DNA from wild-type mice gave only one band of Identifying bona fide targets within the nucleus should now provide 424-bp while that from homozygous KI mice produced 2 bands of 258-bp and important clues to the relationship between this process and aging. 166-bp. The heterozygous mice produced 3 bands of 424-, 258-, and 166-bp.

Materials and Methods Statistical Analysis. Data from image analysis are presented as means Ϯ SE. Statistical comparisons were made using a two-way ANOVA, with P Ͻ 0.05 being Genotyping of Mice. Genomic DNA was isolated from tail fragments by standard considered significant. phenol/chloroform extraction and isopropyl alcohol precipitation. PCR was con- ducted to determine the genotype at the Pthrp locus. DNA was amplified with ACKNOWLEDGMENTS. This work was supported by the Key Project grant (No. forward primer 5Ј-GCTGTGTCTGAACATCAGCTAC-3Ј and reverse primer 5Ј- 30830103) from National Nature and Scientific Foundation of China to D.M., and by ATGCGTCCTTA-AGCTGGGCTC-3Ј. Cycling conditions were 94 °C for 30 sec, 60 °C operating grants from the Canadian Institutes for Health Research to A.C.K. and D.G.

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