Parathyroid Hormone-Related Protein, Its Regulation of Cartilage and Bone Development, and Role in Treating Bone Diseases.

Although parathyroid hormone-related protein (PTHrP) was discovered as a cancer-derived hormone, it has been revealed as an important paracrine/autocrine regulator in many tissues, where its effects are context dependent. Thus its location and action in the vasculature explained decades-long observations that injection of PTH into animals rapidly lowered blood pressure by producing vasodilatation. Its roles have been specified in development and maturity in cartilage and bone as a crucial regulator of endochondral bone formation and bone remodeling, respectively. Although it shares actions with parathyroid hormone (PTH) through the use of their common receptor, PTHR1, PTHrP has other actions mediated by regions within the molecule beyond the amino-terminal sequence that resembles PTH, including the ability to promote placental transfer of calcium from mother to fetus. A striking feature of the physiology of PTHrP is that it possesses structural features that equip it to be transported in and out of the nucleus, and makes use of a specific nuclear import mechanism to do so. Evidence from mouse genetic experiments shows that PTHrP generated locally in bone is essential for normal bone remodeling. Whereas the main physiological function of PTH is the hormonal regulation of calcium metabolism, locally generated PTHrP is the important physiological mediator of bone remodeling postnatally. Thus the use of intermittent injection of PTH as an anabolic therapy for bone appears to be a pharmacological application of the physiological function of PTHrP. There is much current interest in the possibility of developing PTHrP analogs that might enhance the therapeutic anabolic effects.

Involvement of the Parathyroid Hormone-Related Protein on Changes in the CYP3A Expression in Cancer Cachexia.

Parathyroid hormone-related protein (PTHrP), which is secreted from a tumor, contributes to the progression of cachexia, a condition that is observed in half of all cancer patients. Although drug clearance was reported to decrease in patients with cancer cachexia, the details have not been clarified. The present study reports on an investigation of whether PTHrP is involved in the alternation of drug metabolism in cases of cancer cachexia. Cancer cachexia model rats with elevated serum PTHrP levels showed a significant decrease in hepatic and intestinal CYP3A2 protein expression. When midazolam, a CYP3A substrate drug, was administered intravenously or orally to the cancer cachexia rats, its area under the curve (AUC) was increased by about 2 and 5 times, as compared to the control group. Accordingly, the bioavailability of midazolam was increased by about 3 times, thus enhancing its pharmacological effect. In vitro experiments using HepG2 cells and Caco-2 cells showed that the addition of serum from cancer cachexia rats or active PTHrP (1-34) to each cell resulted in a significant decrease in the expression of CYP3A4 mRNA. Treatment with a cell-permeable cAMP analog also resulted in a decreased CYP3A4 expression. Pretreatment with protein kinase A (PKA), protein kinase C (PKC), and nuclear factor-kappa B (NF-κB) inhibitors recovered the decrease in CYP3A4 expression that was induced by PTHrP (1-34). These results suggest that PTHrP suppresses CYP3A expression via the cAMP/PKA/PKC/NF-κB pathway. Therefore, it is likely that PTHrP would be involved in the changes in drug metabolism observed in cancer cachexia.

Wnt16 attenuates osteoarthritis progression through a PCP/JNK-mTORC1-PTHrP cascade.

OBJECTIVES: Wnt16 is implicated in bone fracture and bone mass accrual both in animals and humans. However, its functional roles and molecular mechanism in chondrocyte differentiation and osteoarthritis (OA) pathophysiology remain largely undefined. In this study, we analysed its mechanistic association and functional relationship in OA progression in chondrocyte lineage. METHODS: The role of Wnt16 during skeletal development was examined by Col2a1-Wnt16 transgenic mice and Wnt16fl/fl;Col2a1-Cre (Wnt16-cKO) mice. OA progression was assessed by micro-CT analysis and Osteoarthritis Research Society International score after anterior cruciate ligament transection (ACLT) surgery with Wnt16 manipulation by adenovirus intra-articular injection. The molecular mechanism was investigated in vitro using 3D chondrocyte pellet culture and biochemical analyses. Histological analysis was performed in mouse joints and human cartilage specimens. RESULTS: Wnt16 overexpression in chondrocytes in mice significantly inhibited chondrocyte hypertrophy during skeletal development. Wnt16 deficiency exaggerated OA progression, whereas intra-articular injection of Ad-Wnt16 markedly attenuated ACLT-induced OA. Cellular and molecular analyses showed that, instead of ß-catenin and calcium pathways, Wnt16 activated the planar cell polarity (PCP) and JNK pathway by interacting mainly with AP2b1, and to a lesser extend Ror2 and CD146, and subsequently induced PTHrP expression through phosphor-Raptor mTORC1 pathway. CONCLUSIONS: Our findings indicate that Wnt16 activates PCP/JNK and crosstalks with mTORC1-PTHrP pathway to inhibit chondrocyte hypertrophy. Our preclinical study suggests that Wnt16 may be a potential therapeutic target for OA treatment.

Autocrine parathyroid hormone-like hormone promotes intrahepatic cholangiocarcinoma cell proliferation via increased ERK/JNK-ATF2-cyclinD1 signaling.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is an aggressive tumor with a high fatality rate. It was recently found that parathyroid hormone-like hormone (PTHLH) was frequently overexpressed in ICC compared with non-tumor tissue. This study aimed to elucidate the underlying mechanisms of PTHLH in ICC development. METHODS: The CCK-8 assay, colony formation assays, flow cytometry and a xenograft model were used to examine the role of PTHLH in ICC cells proliferation. Immunohistochemistry (IHC) and western blot assays were used to detect target proteins. Luciferase reporter, chromatin immunoprecipitation (ChIP) and DNA pull-down assays were used to verify the transcription regulation of activating transcription factor-2 (ATF2). RESULTS: PTHLH was significantly upregulated in ICC compared with adjacent and normal tissues. Upregulation of PTHLH indicated a poor pathological differentiation and intrahepatic metastasis. Functional study demonstrated that PTHLH silencing markedly suppressed ICC cells growth, while specific overexpression of PTHLH has the opposite effect. Mechanistically, secreted PTHLH could promote ICC cell growth by activating extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways, and subsequently upregulated ATF2 and cyclinD1 expression. Further study found that the promoter activity of PTHLH were negatively regulated by ATF2, indicating that a negative feedback loop exists. CONCLUSIONS: Our findings demonstrated that the ICC-secreted PTHLH plays a characteristic growth-promoting role through activating the canonical ERK/JNK-ATF2-cyclinD1 signaling pathways in ICC development. We identified a negative feedback loop formed by ATF2 and PTHLH. In this study, we explored the therapeutic implication for ICC patients.

Molecular mechanisms associated with PTHrP-induced proliferation of colon cancer cells.

Parathyroid Hormone-related Protein (PTHrP) is normally produced in many tissues and is recognized for its endocrine, paracrine, autocrine and intracrine modes of action. PTHrP is also implicated in different types of cancer and its expression correlates with the severity of colon carcinoma. Using the human colon cell line Caco-2 we recently obtained evidence that PTHrP, through a paracrine pathway, exerts a protective effect under apoptotic conditions. However, if exogenous PTHrP is able or not to induce the proliferation of these intestinal tumor cells is not known. We found that PTHrP treatment increases the number of live Caco-2 cells. The hormone induces the phosphorylation and nuclear translocation of ERK 1/2, α p38 MAPK, and Akt, without affecting JNK phosphorylation. In addition, PTHrP-dependent ERK phosphorylation is reverted when PI3K activity was inhibited. Following MAPKs nuclear translocation, the transcription factors ATF-1 and CREB were activated in a biphasic manner. In addition PTHrP induces the translocation into the nucleus of ß-catenin, protein that plays key role in maintaining the growth and proliferation of colorectal cancer, and increases the amount of both positive cell cycle regulators c-Myc and Cyclin D. Studies with ERK1/2, α p38 MAPK, and PI3K specific inhibitors showed that PTHrP regulates Caco-2 cell proliferation via these signaling pathways. In conclusion, the results obtained in this work expand our knowledge on the role of exogenous PTHrP in intestinal tumor cells and identify the signaling pathways that are involved in the mitogenic effect of the hormone on Caco-2 cells.

Involvement of ERK1/2, p38 MAPK, and PI3K/Akt signaling pathways in the regulation of cell cycle progression by PTHrP in colon adenocarcinoma cells.

Parathyroid hormone-related peptide (PTHrP) is distributed in most fetal and adult tissues, and its expression correlates with the severity of colon carcinoma. Recently we obtained evidence that in Caco-2 cells, a cell line from human colorectal adenocarcinoma, exogenous PTHrP increases the number of live cells, via ERK1/2, p38 MAPK, and PI3-kinase and induces the expression of cyclin D1, a cell cycle regulatory protein. In this study, we further investigated the role of PTHrP in the regulation of the cell cycle progression in these intestinal cells. Flow cytometry analysis revealed that PTHrP treatment diminishes the number of cells in the G0/G1 phase and increases the number in both S and G2/M phases. The hormone increases the expression of CDK6 and diminishes the amount of negative cell cycle regulators p27Kip1, p15INK4B, and p53. However, PTHrP does not modify the expression of cyclin D3, CDK4, and p16INK4A. In addition, inhibitors of ERK1/2 (PD98059), p38 MAPK (SB203580), and PI3Kinase (LY294002) reversed PTHrP response in Caco-2 cells. Taken together, our results suggest that PTHrP positively modulates cell cycle progression and changes the expression of proteins involved in cell cycle regulation via ERK1/2, p38 MAPK, and PI3K signaling pathways in Caco-2 cells.

Periosteal PTHrP regulates cortical bone modeling during linear growth in mice.

The modeling of long bone surfaces during linear growth is a key developmental process, but its regulation is poorly understood. We report here that parathyroid hormone-related peptide (PTHrP) expressed in the fibrous layer of the periosteum (PO) drives the osteoclastic (OC) resorption that models the metaphyseal-diaphyseal junction (MDJ) in the proximal tibia and fibula during linear growth. PTHrP was conditionally deleted (cKO) in the PO via Scleraxis gene targeting (Scx-Cre). In the lateral tibia, cKO of PTHrP led to a failure of modeling, such that the normal concave MDJ was replaced by a mound-like deformity. This was accompanied by a failure to induce receptor activator of NF-kB ligand (RANKL) and a 75% reduction in OC number (P ≤ 0.001) on the cortical surface. The MDJ also displayed a curious threefold increase in endocortical osteoblast mineral apposition rate (P ≤ 0.001) and a thickened cortex, suggesting some form of coupling of endocortical bone formation to events on the PO surface. Because it fuses distally, the fibula is modeled only proximally and does so at an extraordinary rate, with an anteromedial cortex in CD-1 mice that was so moth-eaten that a clear PO surface could not be identified. The cKO fibula displayed a remarkable phenotype, with a misshapen club-like metaphysis and an enlargement in the 3D size of the entire bone, manifest as a 40-45% increase in the PO circumference at the MDJ (P ≤ 0.001) as well as the mid-diaphysis (P ≤ 0.001). These tibial and fibular phenotypes were reproduced in a Scx-Cre-driven RANKL cKO mouse. We conclude that PTHrP in the fibrous PO mediates the modeling of the MDJ of long bones during linear growth, and that in a highly susceptible system such as the fibula this surface modeling defines the size and shape of the entire bone.

Parathyroid hormone-related peptide and the hair cycle - is it the agonists or the antagonists that cause hair growth?

While the effects of PTHrP have been studied for almost 20 years, most of these studies have focused on effects on the termination of the anagen phase, giving an incomplete picture of the overall effect of PTHrP on the hair cycle. PTHrP was determined in several experimental models to promote transition of hair follicles from anagen to catagen phase, which by itself would suggest that PTHrP blockade might prolong the anagen phase and promote hair growth. However, clinical trials with topically applied PTHrP antagonists have been disappointing, leading to a reconsideration of this model. Additional studies performed in mouse models where hair follicles are damaged (alopecia areata, chemotherapy-induced alopecia) suggest that PTHrP has effects early in the hair cycle as well, promoting hair follicles' entry into anagen phase and initiates the hair cycle. While the mechanism of this has yet to be elucidated, it may involve activation of the Wnt pathway. Thus, the overall effect of PTHrP is to stimulate and accelerate the hair cycle, and in the more clinically relevant models of hair loss where hair follicles have been damaged or become quiescent, it is the agonists, not the antagonists, which would be expected to promote hair growth.

Endocrine regulation of carbonate precipitate formation in marine fish intestine by stanniocalcin and PTHrP.

In marine fish, high epithelial bicarbonate secretion by the intestine generates luminal carbonate precipitates of divalent cations that play a key role in water and ion homeostasis. In vitro studies highlight the involvement of the calciotropic hormones PTHrP (parathyroid hormone-related protein) and stanniocalcin (STC) in the regulation of epithelial bicarbonate transport. The present study tested the hypothesis that calciotropic hormones have a regulatory role in carbonate precipitate formation in vivo. Sea bream (Sparus aurata) juveniles received single intraperitoneal injections of piscine PTHrP(1-34), the PTH/PTHrP receptor antagonist PTHrP(7-34) or purified sea bream STC, or were passively immunized with polyclonal rabbit antisera raised against sea bream STC (STC-Ab). Endocrine effects on the expression of the basolateral sodium bicarbonate co-transporter (Slc4a4.A), the apical anion exchangers Slc26a6.A and Slc26a3.B, and the V-type proton pump ß-subunit (Atp6v1b) in the anterior intestine were evaluated. In keeping with their calciotropic nature, the hypocalcaemic factors PTHrP(7-34) and STC up-regulated gene expression of all transporters. In contrast, the hypercalcaemic factor PTHrP(1-34) and STC antibodies down-regulated transporters involved in the bicarbonate secretion cascade. Changes in intestine luminal precipitate contents provoked by calcaemic endocrine factors validated these results: 24 h post-injection either PTHrP(1-34) or immunization with STC-Ab reduced the carbonate precipitate content in the sea bream intestine. In contrast, the PTH/PTHrP receptor antagonist PTHrP(7-34) increased not only the precipitated fraction but also the concentration of HCO3(-) equivalents in the intestinal fluid. These results confirm the hypothesis that calciotropic hormones have a regulatory role in carbonate precipitate formation in vivo in the intestine of marine fish. Furthermore, they illustrate for the first time in fish the counteracting effect of PTHrP and STC, and reveal an unexpected contribution of calcaemic factors to acid-base balance.

[Role for PTHrP in bone and cartilage metabolism].

PTH-related peptide (PTHrP) is a widely distributed cytokine, which shares the cognate receptor PTHR1 with PTH. Originally identified as a causal factor of humoral hypercalcemia of malignancy twenty years ago, PTHrP is now recognized as a critical physiological regulator of various biological processes, including bone and cartilage metabolism.

Src kinases mediate VEGFR2 transactivation by the osteostatin domain of PTHrP to modulate osteoblastic function.

Parathyroid hormone-related protein (PTHrP) stimulates osteoblastic function through its N- and C-terminal domains. Since the osteogenic action of the latter domain appears to depend at least in part on its interaction with the vascular endothelial growth factor (VEGF) system, we aimed to explore the putative mechanism underlying this interaction in osteoblasts. Using native conditions for protein extraction and immunoblotting, we found that both PTHrP (107-139) and the shorter PTHrP (107-111) peptide (known as osteostatin), at 100 nM, promoted the appearance of a VEGF receptor (VEGFR) 2 protein band of apparent Mr. wt. 230 kDa, which likely represents its activation by dimer formation, in mouse osteoblastic MC3T3-E1 cells. Moreover, osteostatin (100 nM) maximally increased VEGFR2 phosphorylation at Tyr-1059 within 5-10 min in both MC3T3-E1 and rat osteoblastic osteosarcoma UMR-106 cells. This phosphorylation elicited by osteostatin appears to be VEGF-independent, but prevented by the VEGFR2 activation inhibitor SU1498 and also by the Src kinase inhibitors SU6656 and PP1. Furthermore, osteostatin induced phosphorylation of Src, extracellular signal-regulated kinase (ERK) and Akt with a similar time course to that observed for VEGFR2 activation in these osteoblastic cells. This osteostatin-dependent induction of ERK and Akt activation was abrogated by SU6656. Up-regulation of VEGF and osteoprotegerin gene expression as well as the pro-survival effect induced by osteostatin treatment were all prevented by both SU1498 and SU6656 in these osteoblastic cells. Collectively, these findings demonstrate that the osteostatin domain of C-terminal PTHrP phosphorylates VEGFR2 through Src activation, which represents a mechanism for modulating osteoblastic function.

Knockdown of parathyroid hormone related protein in smooth muscle cells alters renal hemodynamics but not blood pressure.

Parathyroid hormone-related protein (PTHrP) belongs to vasoactive factors that regulate blood pressure and renal hemodynamics both by reducing vascular tone and raising renin release. PTHrP is expressed in systemic and renal vasculature. Here, we wanted to assess the contribution of vascular smooth muscle cell endogenous PTHrP to the regulation of cardiovascular and renal functions. We generated a mouse strain (SMA-CreERT2/PTHrPL2/L2 or premutant PTHrPSM-/-), which allows temporally controlled, smooth muscle-targeted PTHrP knockdown in adult mice. Tamoxifen treatment induced efficient recombination of PTHrP-floxed alleles and decreased PTHrP expression in vascular and visceral smooth muscle cells of PTHrPSM-/- mice. Blood pressure remained unchanged in PTHrPSM-/- mice, but plasma renin concentration and creatinine clearance were reduced. Renal hemodynamics were further analyzed during clearance measurements in anesthetized mice. Conditional knockdown of PTHrP decreased renal plasma flow and glomerular filtration rate with concomitant reduction in filtration fraction. Similar measurements were repeated during acute saline volume expansion. Saline volume expansion induced a rise in renal plasma flow and reduced filtration fraction; both were blunted in PTHrPSM-/- mice leading to impaired diuresis. These findings show that endogenous vascular smooth muscle PTHrP controls renal hemodynamics under basal conditions, and it is an essential factor in renal vasodilation elicited by saline volume expansion.

Genes associate with abnormal bone cell activity in bone metastasis.

Bone is one of the most frequent sites of metastasis in patients with malignancies. Up to 90 % of patients with multiple myeloma, and 60 % to 75 % patients with prostate cancer and breast cancer develop bone metastasis at the later stages of their diseases. Bone metastases are responsible for tremendous morbidity in patients with cancer, including severe bone pain, pathologic fractures, spinal cord and nerve compression syndromes, life-threatening hypercalcemia, and increased mortality. Multiple factors produced by tumor cells or produced by the bone marrow microenvironment in response to tumor cells play important roles in activation of osteoclastic bone resorption and modulation of osteoblastic activity in patients with bone metastasis. In this chapter, we will review the genes that play important roles in bone destruction, tumor growth, and osteoblast activity in bone metastasis and discuss the potential therapies targeting the products of these genes to block both bone destruction and tumor growth.

Alterations in phosphorus, calcium and PTHrP contribute to defects in dental and dental alveolar bone formation in calcium-sensing receptor-deficient mice.

To determine whether the calcium-sensing receptor (CaR) participates in tooth formation and dental alveolar bone development in mandibles in vivo, we examined these processes, as well as mineralization, in 2-week-old CaR-knockout (CaR(-/-)) mice. We also attempted to rescue the phenotype of CaR(-/-) mice by genetic means, in mice doubly homozygous for CaR and 25-hydroxyvitamin D 1alpha-hydroxylase [1alpha(OH)ase] or parathyroid hormone (Pth). In CaR(-/-) mice, which exhibited hypercalcemia, hypophosphatemia and increased serum PTH, the volumes of teeth and of dental alveolar bone were decreased dramatically, whereas the ratio of the area of predentin to total dentin and the number and surface of osteoblasts in dental alveolar bone were increased significantly, as compared with wild-type littermates. The normocalcemia present in CaR(-/-);1alpha(OH)ase(-/-) mice only slightly improved the defects in dental and alveolar bone formation observed in the hypercalcemic CaR(-/-) mice. However, these defects were completely rescued by the additional elimination of hypophosphatemia and by an increase in parathyroid hormone-related protein (PTHrP) expression in the apical pulp, Hertwig's epithelial root sheath and mandibular tissue in CaR(-/-); Pth(-/-) mice. Therefore, alterations in calcium, phosphorus and PTHrP contribute to defects in the formation of teeth and alveolar bone in CaR-deficient mice. This study indicates that CaR participates in the formation of teeth and in the development of dental alveolar bone in mandibles in vivo, although it appears to do so largely indirectly.

New tools for studying osteoarthritis genetics in zebrafish.

OBJECTIVE: Increasing evidence points to a strong genetic component to osteoarthritis (OA) and that certain changes that occur in osteoarthritic cartilage recapitulate the developmental process of endochondral ossification. As zebrafish are a well validated model for genetic studies and developmental biology, our objective was to establish the spatiotemporal expression pattern of a number of OA susceptibility genes in the larval zebrafish providing a platform for functional studies into the role of these genes in OA. DESIGN: We identified the zebrafish homologues for Mcf2l, Gdf5, PthrP/Pthlh, Col9a2, and Col10a1 from the Ensembl genome browser. Labelled probes were generated for these genes and in situ hybridisations were performed on wild type zebrafish larvae. In addition, we generated transgenic reporter lines by modification of bacterial artificial chromosomes (BACs) containing full length promoters for col2a1 and col10a1. RESULTS: For the first time, we show the spatiotemporal expression pattern of Mcf2l. Furthermore, we show that all six putative OA genes are dynamically expressed during zebrafish larval development, and that all are expressed in the developing skeletal system. Furthermore, we demonstrate that the transgenic reporters we have generated for col2a1 and col10a1 can be used to visualise chondrocyte hypertrophy in vivo. CONCLUSION: In this study we describe the expression pattern of six OA susceptibility genes in zebrafish larvae and the generation of two new transgenic lines marking chondrocytes at different stages of maturation. Moreover, the tools used demonstrate the utility of the zebrafish model for functional studies on genes identified as playing a role in OA.

Parathyroid hormone-related protein is induced by hypoxia and promotes expression of the differentiated phenotype of human articular chondrocytes.

PTHrP (parathyroid hormone-related protein) is crucial for normal cartilage development and long bone growth and acts to delay chondrocyte hypertrophy and terminal differentiation in the growth plate. After growth plate closure adult HACs (human articular chondrocytes) still produce PTHrP, suggesting a possible role for this factor in the permanent articular cartilage. However, the expression regulation and function of PTHrP in the permanent articular cartilage is unknown. Human articular cartilage is an avascular tissue and functions in a hypoxic environment. The resident chondrocytes have adapted to hypoxia and use it to drive their tissue-specific functions. In the present study, we explored directly in normal articular chondrocytes isolated from a range of human donors the effect of hypoxia on PTHrP expression and whether PTHrP can regulate the expression of the permanent articular chondrocyte phenotype. We show that in HACs PTHrP is up-regulated by hypoxia in a HIF (hypoxia-inducible factor)-1α and HIF-2α-dependent manner. Using recombinant PTHrP, siRNA-mediated depletion of endogenous PTHrP and by blocking signalling through its receptor [PTHR1 (PTHrP receptor 1)], we show that hypoxia-induced PTHrP is a positive regulator of the key cartilage transcription factor SOX9 [SRY (sex determining region on the Y chromosome)-box 9], leading to increased COL2A1 (collagen type II, α1) expression. Our findings thus identify PTHrP as a potential factor for cartilage repair therapies through its ability to promote the differentiated HAC phenotype.

[Identification of osteogenic signal and the development of artificial bones].

Mammalian skeletons are formed through two distinct processes, intramembranous ossification and endochondral ossification. During the endochondral ossification, indian hedgehog (Ihh) /parathyroid hormone-related protein (PTHrP) negative-feedback loop regulates the distance between epiphysis and the layer of hypertrophic chondrocytes. Ihh also stimulates bone formation. Based on such knowledge, bioactive molecule-loaded artificial bones are expected to achieve efficient bone regeneration without cell transplantation.

Bone metastases: molecular mechanisms and novel therapeutic interventions.

It has been long recognized that skeleton represents one of the most favored metastatic sites for common cancers like breast and prostate. During the last decade the molecular mechanisms that are responsible for the development of bone metastasis have been gradually illuminated. It appears that the bone microenvironment has a pivotal role in this process. Metastatic tumor cells interact with bone triggering a cascade of molecular events that produce osteolytic and/or osteoblastic phenomena. In this review, we summarize and discuss the most significant factors and signaling pathways implicated in bone colonization. Moreover, based on the recent literature and data, we foresee the need for designing novel agents that will efficiently disrupt these interactions among cancer cells and bone microenvironment, bringing hope for more effective treatments.

Molecular basis for specificity of nuclear import and prediction of nuclear localization.

Although proteins are translated on cytoplasmic ribosomes, many of these proteins play essential roles in the nucleus, mediating key cellular processes including but not limited to DNA replication and repair as well as transcription and RNA processing. Thus, understanding how these critical nuclear proteins are accurately targeted to the nucleus is of paramount importance in biology. Interaction and structural studies in the recent years have jointly revealed some general rules on the specificity determinants of the recognition of nuclear targeting signals by their specific receptors, at least for two nuclear import pathways: (i) the classical pathway, which involves the classical nuclear localization sequences (cNLSs) and the receptors importin-α/karyopherin-α and importin-ß/karyopherin-ß1; and (ii) the karyopherin-ß2 pathway, which employs the proline-tyrosine (PY)-NLSs and the receptor transportin-1/karyopherin-ß2. The understanding of specificity rules allows the prediction of protein nuclear localization. We review the current understanding of the molecular determinants of the specificity of nuclear import, focusing on the importin-α•cargo recognition, as well as the currently available databases and predictive tools relevant to nuclear localization. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.