Parathyroid hormone-related protein promotes quiescence and survival of serum-deprived chondrocytes by inhibiting rRNA synthesis.

Parathyroid hormone-related protein (PTHrP) was initially recognized for its ability to promote parathyroid hormone-like bioactivity in kidney, bone, and squamous epithelial cells. PTHrP is a multifunctional protein in which bioactivity is mediated by two distinct pathways. Its classic parathyroid hormone-like activity results from binding of its amino terminus to cell surface PTH1R and activation of signal transduction pathways. Another less well recognized pathway involves translocation of PTHrP to the nucleus via a mid-region bipartite nuclear targeting sequence (NTS), similar in structure and function to those found in retroviral regulatory proteins. PTHrP was identified in the nucleus of several different cell types in vivo and in vitro, where it has been implicated in cell cycle progression, cellular differentiation, and apoptosis. In previous work we showed that nuclear translocation of PTHrP enhanced the survival of serum-deprived chondrogenic cells, associated with RNA, and localized to a region of the nucleus rich in complexes of newly transcribed ribosomal RNA and protein. In this work we have used two chondrogenic cell lines, CFK2 (PTH1R+) and 27m21 (PTH1R-) to further explore mechanisms whereby PTHrP rescues immature chondrocytes from apoptosis. Endogenous PTHrP and exogenous PTHrP NTS peptide protected serum-deprived cells from apoptosis, in the presence and absence of PTH1R. The survival of cells expressing PTHrP and those treated with PTHrP NTS peptide was associated with a rapid shift into G(o)/G1 accompanied by a significant down-regulation of rRNA synthesis and a decrease in the number of actively translating polyribosome complexes. Together with our previous observations, this work predicts a role for PTHrP in modulating ribosome biogenesis and preventing chondrogenic cells from progressing through the cell cycle in an unfavorable environment.

Expression of FGFR3 with the G380R achondroplasia mutation inhibits proliferation and maturation of CFK2 chondrocytic cells.

A G380R substitution in the transmembrane-spanning region of FGFR3 (FGFR3Ach) results in constitutive receptor kinase activity and is the most common cause of achondroplastic dwarfism in humans. The epiphyseal growth plates of affected individuals are disorganized and hypocellular and show aberrant chondrocyte maturation. To examine the molecular basis of these abnormalities, we used a chondrocytic cell line, CFK2, to stably express the b variant of wild-type FGFR3 or the the constitutively active FGFR3Ach. Overexpression of FGFR3 had minimal effects on CFK2 proliferation and maturation compared with the severe growth retardation found in cells expressing FGFR3Ach. Cells expressing the mutant receptor also showed an abnormal apoptotic response to serum deprivation and failed to undergo differentiation under appropriate culture conditions. These changes were associated with altered expression of integrin subunits, which effectively led to a switch in substrate preference of the immature cell from fibronectin to type II collagen. These in vitro observations support those from in vivo studies indicating that FGFR3 mediates an inhibitory influence on chondrocyte proliferation. We now suggest that the mechanism is related to altered integrin expression.

The nucleolar targeting signal (NTS) of parathyroid hormone related protein mediates endocytosis and nucleolar translocation.

Previous work has identified the parathyroid hormone-related protein (PTHrP) nucleolar targeting signal (NTS) as both necessary and sufficient for localization of PTHrP to the nucleus and nucleolus of a variety of cells where it is believed to participate in the regulation of cell proliferation, differentiation, and apoptotic cell death. The mechanism whereby a secreted peptide, such as PTHrP, gains access to the nuclear compartment remains a question of debate. The current work examines the possibility that exogenous PTHrP is internalized and transported to the nuclear compartment by a mechanism that is dependent on preservation of the PTHrP NTS. Transiently expressed, PTHrP(1-141) was detected at the cell surface as well as in the cytoplasmic and nuclear compartments of COS-1 cells. Deletion of the NTS, or mutation of the conserved GxKKxxK motif within the NTS, effectively prevented both cell-surface binding and nuclear/nucleolar accumulation of PTHrP(1-141). A biotinylated peptide corresponding to the PTHrP NTS (PTHrP-NTS-biotin) was internalized and translocated to the nucleus and nucleolus in a time-, temperature-, and concentration-dependent manner, whereas a peptide representing a similar bipartite NTS from Nucleolin was not. Internalization and nucleolar targeting of PTHrP-NTS-biotin were indistinguishable in CFK2 cells, which express the common PTH/PTHrP receptor, and in 27m21 cells, which do not. In addition, pretreatment with a saturating dose of synthetic PTHrP(74-113) was capable of abrogating nucleolar accumulation of the PTHrP-NTS peptide, whereas pretreatment with PTHrP(1-34) or PTHrP(67-86) was not. These observations demonstrate that binding of exogenous, full-length PTHrP to the cell surface is mediated through a conserved motif embedded in the NTS and suggest that internalization and nucleolar targeting of an NTS peptide are mediated through binding to a cell surface protein distinct from the PTH/PTHrP receptor. In total, the data support the hypothesis that secreted PTHrP(1-141) can be endocytosed and targeted to the nucleolus through a mechanism that is dependent on preservation of a core motif within the PTHrP NTS.

Parathyroid hormone-related protein interacts with RNA.

Parathyroid hormone-related protein (PTHrP) is a secreted protein that acts as an autocrine and paracrine mediator of cell proliferation and differentiation. In addition to its biological activity that is mediated through signal transduction cascades, there is evidence for an intracellular role for PTHrP in cell cycle progression and apoptosis. These effects are mediated through a mid-region nuclear targeting sequence (NTS) that localizes PTHrP to the region of the nucleolus where ribonucleoprotein complexes form in vivo. In this work, we show that endogenous, transfected, and in vitro translated PTHrP proteins bind homopolymeric and total cellular RNAs at salt concentrations up to 1 M. A peptide representing the PTHrP NTS was effective in competing with the wild-type protein for RNA binding, whereas a similar peptide representing the nucleolin NTS was not. Site-directed mutagenesis revealed that the binding of PTHrP to RNA was direct and was dependent on preservation of a core GXKKXXK motif, embedded in the PTHrP NTS, which is shared with other RNA-binding proteins. The current observations are the first to document RNA binding by a secreted cellular protein and predict a role for PTHrP in regulating RNA metabolism that may be related to its localization in the nucleolus of cells in vivo.

E-cadherin and calretinin: a useful combination of immunochemical markers for differentiation between mesothelioma and metastatic adenocarcinoma.

AIMS: To assess the diagnostic accuracy of combinations of antibodies in the differential diagnosis of metastatic carcinomas and mesotheliomas in pleural lesions. METHODS AND RESULTS: The specificity and sensitivity of the commercially available antibodies Ber-EP4, MOC-31, CEA, B72.3, CD15, E-cadherin and calretinin were evaluated using an indirect immunoperoxidase staining technique. This technique was applied to formalin-fixed, paraffin-embedded tissue blocks of pleural lesions. Twenty-one patients with metastatic carcinoma (MC) and 20 patients with malignant mesothelioma (MM) were included. The combination E-cadherin/calretinin had the highest specificity (MC 100% and MM 91%) and sensitivity (MM 100% and MC 91%) considering both categories of tumours. CONCLUSIONS: The combination of E-cadherin/calretinin appears to be a suitable panel for distinguishing metastatic carcinomas and mesotheliomas in pleural lesions. The additional value of other markers is limited.