The functions of parathyroid hormone-related protein (PTHrP) on morphogenesis, cell proliferation, apoptosis, and calcium homeostasis have been attributed to its N terminus. Evidence suggests that many of these effects are not mediated by the N terminus but by the midregion, a nuclear localization sequence (NLS), and C terminus of the protein. A knock-in mouse lacking the midregion, NLS, and C terminus of PTHrP (Pthrp(Delta/Delta)) was developed. Pthrp(Delta/Delta) mice had craniofacial dysplasia, chondrodysplasia, and kyphosis, with most mice dying by d 5 of age. In bone, there were fewer chondrocytes and osteoblasts per area, bone mass was decreased, and the marrow was less cellular, with erythroid hypoplasia. Cellular proliferation was impaired, and apoptosis was increased. Runx2, Ocn, Sox9, Crtl1, beta-catenin, Runx1, ephrin B2, cyclin D1, and Gata1 were underexpressed while P16/Ink4a, P21, GSK-3beta, Il-6, Ffg3, and Ihh were overexpressed. Mammary gland development was aberrant, and energy metabolism was deregulated. These results establish that the midregion, NLS, and C terminus of PTHrP are crucial for the commitment of osteogenic and hematopoietic precursors to their lineages, and for survival, and many of the effects of PTHrP on development are not mediated by its N terminus. The down-regulation of Runx1, Runx2, and Sox9 indicates that PTHrP is a modulator of transcriptional activation during stem cell commitment.

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Evidence exists that parathyroid hormone-related protein (PTHrP) 1-34 may be more anabolic in bone than parathyroid hormone (PTH) 1-34. While optical imaging is growing in popularity, scant information exists on the relationships between traditional bone imaging and histology and bioluminescence (BLI) and fluorescence (FLI) imaging. We aimed to evaluate the effects of PTHrP 1-34 on bone mass and determine if relationships existed between radiographic and histologic findings in bone and BLI and FLI indices. Vertebrae (vossicles) from mice coexpressing luciferase and green fluorescent protein were implanted subcutaneously into allogenic nude mice. Transplant recipients were treated daily with saline or PTHrP 1-34 for 4 weeks. BLI, FLI, radiography, histology, and μCT of the vossicles were performed over time. PTHrP 1-34 increased bioluminescence the most after 2 weeks, fluorescence at all time points, and decreased the time to peak bioluminescence at 4 weeks (P ≤ 0.027), the latter of which suggesting enhanced engraftment. PTHrP 1-34 maximized vertebral body volume at 4 weeks (P < 0.0001). The total amount of bone observed histologically increased in both groups at 2 and 4 weeks (P ≤ 0.002); however, PTHrP 1-34 exceeded time-matched controls (P ≤ 0.044). A positive linear relationship existed between the percentage of trabecular bone and 1) total bioluminescence (r = 0.595; P = 0.019); 2) total fluorescence (r = 0.474; P = 0.074); and 3) max fluorescence (r = 0.587; P = 0.021). In conclusion, PTHrP 1-34 enhances engraftment and bone mass, which can be monitored non-invasively by BLI and FLI.

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The N-terminus of parathyroid hormone-related protein regulates bone marrow stromal cell differentiation. We hypothesized that the nuclear localization sequence and C-terminus are involved. MicroRNA and gene expression analyses were performed on bone marrow stromal cells from mice lacking the nuclear localization sequence and C-terminus (Pthrp Δ/Δ ) and age-matched controls. Differentiation assays with microRNA, cytochemical/histologic/morphologic, protein, and gene expression analyses were performed. Pthrp Δ/Δ bone marrow stromal cells are anti-osteochondrogenic, pro-adipogenic, and pro-myogenic, expressing more Klf4, Gsk-3β, Lif, Ct-1, and microRNA-434 but less β-catenin, Igf-1, Taz, Osm, and microRNA-22 (p ⩽ 0.024). Pthrp Δ/Δ osteoblasts had less mineralization, osteocalcin, Runx2, Osx, Igf-1, and leptin (p ⩽ 0.029). Pthrp Δ/Δ produced more adipocytes, Pparγ, and aP2, but less Lpl (p ⩽ 0.042). Pthrp Δ/Δ cartilage pellets were smaller with less Sox9 and Pth1r, but greater Col2a1 (p ⩽ 0.024). Pthrp Δ/Δ produced more myocytes, Des, and Myog (p ⩽ 0.021). MicroRNA changes supported these findings. In conclusion, the nuclear localization sequence and C-terminus are pro-osteochondrogenic, anti-adipogenic, and anti-myogenic.