Activating Inducible T-cell Costimulator Yields Antitumor Activity Alone and in Combination with Anti-PD-1 Checkpoint Blockade.

In recent years, there has been considerable interest in mAb-based induction of costimulatory receptor signaling as an approach to combat cancer. However, promising nonclinical data have yet to translate to a meaningful clinical benefit. Inducible T-cell costimulator (ICOS) is a costimulatory receptor important for immune responses. Using a novel clinical-stage anti-ICOS immunoglobulin G4 mAb (feladilimab), which induces but does not deplete ICOS+ T cells and their rodent analogs, we provide an end-to-end evaluation of the antitumor potential of antibody-mediated ICOS costimulation alone and in combination with programmed cell death protein 1 (PD-1) blockade. We demonstrate, consistently, that ICOS is expressed in a range of cancers, and its induction can stimulate growth of antitumor reactive T cells. Furthermore, feladilimab, alone and with a PD-1 inhibitor, induced antitumor activity in mouse and humanized tumor models. In addition to nonclinical evaluation, we present three patient case studies from a first-time-in-human, phase I, open-label, dose-escalation and dose-expansion clinical trial (INDUCE-1; ClinicalTrials.gov: NCT02723955), evaluating feladilimab alone and in combination with pembrolizumab in patients with advanced solid tumors. Preliminary data showing clinical benefit in patients with cancer treated with feladilimab alone or in combination with pembrolizumab was reported previously; with example cases described here. Additional work is needed to further validate the translation to the clinic, which includes identifying select patient populations that will benefit from this therapeutic approach, and randomized data with survival endpoints to illustrate its potential, similar to that shown with CTLA-4 and PD-1 blocking antibodies. Significance: Stimulation of the T-cell activation marker ICOS with the anti-ICOS agonist mAb feladilimab, alone and in combination with PD-1 inhibition, induces antitumor activity across nonclinical models as well as select patients with advanced solid tumors.

A human CD137×PD-L1 bispecific antibody promotes anti-tumor immunity via context-dependent T cell costimulation and checkpoint blockade.

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.

Effect of phosphorus and potassium addition on greenhouse gas emissions and nutrient utilization of a rice-fish co-culture system.

Greenhouse gas (GHG) emissions from aquaculture have gained widespread attention. However, the effect of phosphorus (P) and potassium (K) on GHG emissions from aquaculture systems has rarely been studied. In this study, we conducted a laboratory-scale experiment to investigate the effect of P and K addition on CH4 and N2O emissions and nutrient use efficiency in a rice-fish co-culture system. The results showed that the CH4 flux rate did not differ between the rice-fish co-culture (RF) and fish monoculture (F) systems. Phosphorus addition did not affect CH4 emission from the RF. In contrast, K addition significantly increased the CH4 emission from the RF by 148.4%. Dual P and K addition greatly increased the CH4 emission from the RF by six times, indicating an interactive effect of P and K on the stimulation of CH4 emission. Phosphorus addition strengthened the restorative effect of the RF on N2O emission, while K addition weakened the restorative effect of the RF on N2O emission. The combination of P and K did not affect the N2O emission from the RF. The application of P and K strengthened the restorative effect of rice on nitrogen (N) pollution in aquaculture water. Phosphorus and K addition significantly increased the rice biomass and nutrient in the harvested rice, but did not affect the fish biomass and nutrient in the harvested fish. Dual P and K addition increased the nutrient use efficiency in the rice-fish system. These results provide a reference for adjusting nutrient management to reduce GHG emissions and improve nutrient use efficiency in the rice-fish system.

Regulation of Phosphoribosyl-Linked Serine Ubiquitination by Deubiquitinases DupA and DupB.

The family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection.

Spectrum of SLC20A2, PDGFRB, PDGFB, and XPR1 mutations in a large cohort of patients with primary familial brain calcification.

Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder with four causative genes (SLC20A2, PDGFRB, PDGFB, and XPR1) that have been identified. Here, we aim to describe the mutational spectrum of four causative genes in a series of 226 unrelated Chinese PFBC patients. Mutations in four causative genes were detected in 16.8% (38/226) of PFBC patients. SLC20A2 mutations accounted for 14.2% (32/226) of all patients. Mutations in the other three genes were relatively rare, accounting for 0.9% (2/226) of all patients, respectively. Clinically, 44.8% of genetically confirmed patients (probands and relatives) were considered symptomatic. The most frequent symptoms were chronic headache, followed by movement disorders and vertigo. Moreover, the total calcification score was significantly higher in the symptomatic group compared to the asymptomatic group. Functionally, we observed impaired phosphate transport induced by seven novel missense mutations in SLC20A2 and two novel mutations in XPR1. The mutation p.D164Y in XPR1 might result in low protein expression through an enhanced proteasome pathway. In conclusion, our study further confirms that mutations in SLC20A2 are the major cause of PFBC and provides additional evidence for the crucial roles of phosphate transport impairment in the pathogenies of PFBC.

Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination.

Conventional ubiquitination regulates key cellular processes by catalysing the ATP-dependent formation of an isopeptide bond between ubiquitin (Ub) and primary amines in substrate proteins 1 . Recently, the SidE family of bacterial effector proteins (SdeA, SdeB, SdeC and SidE) from pathogenic Legionella pneumophila were shown to use NAD+ to mediate phosphoribosyl-linked ubiquitination of serine residues in host proteins2, 3. However, the molecular architecture of the catalytic platform that enables this complex multistep process remains unknown. Here we describe the structure of the catalytic core of SdeA, comprising mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains, and shed light on the activity of two distinct catalytic sites for serine ubiquitination. The mART catalytic site is composed of an α-helical lobe (AHL) that, together with the mART core, creates a chamber for NAD+ binding and ADP-ribosylation of ubiquitin. The catalytic site in the PDE domain cleaves ADP-ribosylated ubiquitin to phosphoribosyl ubiquitin (PR-Ub) and mediates a two-step PR-Ub transfer reaction: first to a catalytic histidine 277 (forming a transient SdeA H277-PR-Ub intermediate) and subsequently to a serine residue in host proteins. Structural analysis revealed a substrate binding cleft in the PDE domain, juxtaposed with the catalytic site, that is essential for positioning serines for ubiquitination. Using degenerate substrate peptides and newly identified ubiquitination sites in RTN4B, we show that disordered polypeptides with hydrophobic residues surrounding the target serine residues are preferred substrates for SdeA ubiquitination. Infection studies with L. pneumophila expressing substrate-binding mutants of SdeA revealed that substrate ubiquitination, rather than modification of the cellular ubiquitin pool, determines the pathophysiological effect of SdeA during acute bacterial infection.

Diagnosis of Vitreoretinal Aspergillosis with Transvitreal Retinochoroidal Biopsy.

Diagnosing culture-proven endophthalmitis is complicated by the insufficient yield of intraocular samples and the variety of etiologies which mimic true endophthalmitis. In cases of impending vision loss where vitreous biopsy cannot provide a definitive diagnosis, transvitreal retinochoroidal biopsy can be an effective next step. Our case is a 48-year-old male with B-cell acute lymphoblastic leukemia that presented with counting fingers vision, redness, and tearing of the left eye. Exam showed cell and flare with hypopyon as well as dense vitritis. The patient underwent diagnostic pars plana vitrectomy and vitreous culture was negative at the time. Flow cytometry demonstrated no malignant cells. However, the patient's vision and mental status continued to clinically decline despite being started on intravitreal and systemic antibiotic and antifungal therapy. Neuroimaging revealed rim-enhancing brain lesions. Transvitreal retinochoroidal biopsy was performed in an elevated area of the retina. The biopsy helped rule out malignancy and showed acute-angle, septate, branching hyphae characteristic of Aspergillus fumigatus. Ultimately, the vitreous biopsy, cultures, and a biopsy from the left frontal lobe brain abscess all confirmed this diagnosis as well. Transvitreal retinochoroidal biopsy can play a role in the diagnosis of a case of posterior uveitis and can be particularly effective in diagnosing a fungal endophthalmitis.

Analysis of gene expression and functional characterization of XPR1: a pathogenic gene for primary familial brain calcification.

Primary familial brain calcification (PFBC) is a neuropsychiatric disorder characterized by bilateral cerebral calcification with diverse neurologic or psychiatric symptoms. Recently, XPR1 variation has accounted for PFBC as another new causative gene. However, little is known about the distribution and basic function of XPR1 and its interaction with the other three pathogenic genes for PFBC (SLC20A2, PDGFRB and PDGFB). The aim of this study was to further clarify the role of XPR1 in PFBC brain pathology. As a result, gene expression profiles showed that XPR1 mRNA was widely expressed throughout the mouse brain. Cerebellum and striatum, most commonly affected in PFBC, contained a higher level of XPR1 protein than other brain regions. Additionally, XPR1 deficiency seriously affected Pi efflux and XPR1 mutations seemed to have an effect through haploinsufficiency mechanism. The immunoprecipitation and immunohistochemical studies demonstrated that XPR1 could interact with PDGFRB and might form a complex on the cell membrane. These results suggested that XPR1 played a fundamental role in the maintenance of cellular phosphate balance in the brain. This provided us with a novel perspective on understanding the pathophysiology of PFBC. The expression networks and interaction with the known pathogenic genes could shed new light on additional candidate genes for PFBC.

Phosphoribosylation of Ubiquitin Promotes Serine Ubiquitination and Impairs Conventional Ubiquitination.

Conventional ubiquitination involves the ATP-dependent formation of amide bonds between the ubiquitin C terminus and primary amines in substrate proteins. Recently, SdeA, an effector protein of pathogenic Legionella pneumophila, was shown to mediate NAD-dependent and ATP-independent ubiquitin transfer to host proteins. Here, we identify a phosphodiesterase domain in SdeA that efficiently catalyzes phosphoribosylation of ubiquitin on a specific arginine via an ADP-ribose-ubiquitin intermediate. SdeA also catalyzes a chemically and structurally distinct type of substrate ubiquitination by conjugating phosphoribosylated ubiquitin to serine residues of protein substrates via a phosphodiester bond. Furthermore, phosphoribosylation of ubiquitin prevents activation of E1 and E2 enzymes of the conventional ubiquitination cascade, thereby impairing numerous cellular processes including mitophagy, TNF signaling, and proteasomal degradation. We propose that phosphoribosylation of ubiquitin potently modulates ubiquitin functions in mammalian cells.

Elimination of ALDH+ breast tumor initiating cells by docosahexanoic acid and/or gamma tocotrienol through SHP-1 inhibition of Stat3 signaling.

Study investigated the ability of docosahexaenoic acid (DHA) alone and in combination with gamma-tocotrienol (γT3) to eliminate aldehyde dehydrogenase positive (ALDH+) cells and to inhibit mammosphere formation, biomarker and functional assay for tumor initiating cells (TICs), respectively, in human triple negative breast cancer cells (TNBCs), and investigated possible mechanisms of action. DHA upregulated Src homology region 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) protein levels and suppressed levels of phosphorylated signal transducer and activator of transcription-3 (pStat3) and its downstream mediators c-Myc, and cyclin D1. siRNA to SHP-1 enhanced the percentage of ALDH+ cells and Stat-3 signaling, as well as inhibited, in part, the ability of DHA to reduce the percentage of ALDH+ cells and Stat-3 signaling. γT3 alone and in combination with DHA reduced ALDH+ TNBCs, up-regulated SHP-1 protein levels, and suppressed Stat-3 signaling. Taken together, data demonstrate the anti-TIC potential of achievable concentrations of DHA alone as well as in combination with γT3.

A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function.

The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains and that thalidomide blocks the formation of CRBN-ubiquitin conjugates. Furthermore, we show that ubiquitinated CRBN is targeted for proteasomal degradation. Treatment of human myeloma cell lines such as MM1.S, OPM2, and U266 with thalidomide (100 µM) and its structural analog lenalidomide (10 µM) results in stabilization of CRBN and elevation of CRBN protein levels. This in turn leads to the reduced level of CRBN target proteins and enhances the sensitivity of human multiple myeloma cells to IMiDs. Our results reveal a novel mechanism by which thalidomide and its analogs modulate the CRBN function in cells. Through inhibition of CRBN ubiquitination, thalidomide and its analogs allow CRBN to accumulate, leading to the increased cullin-4 RING E3 ligase-mediated degradation of target proteins.

Transcription factor IKZF1 is degraded during the apoptosis of multiple myeloma cells induced by kinase inhibition.

Immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide exhibit high responsive rates for newly identified or relapsed multiple myeloma patients. However, their mechanisms of action are not completely understood. One mechanism involves the ubiquitination and degradation of two transcription factors, IKZF1 and IKZF3. Whether there are other degradation pathways for IKZF1 in myeloma cells remains unknown. Here, we found that although IKZF1 ubiquitination was reduced, its stability was also significantly reduced in MM1.S and OPM2 cells treated with kinase inhibitors, 5,6-dichlorobenzimidazole riboside (DRB) or roscovitine. Through pharmacological inhibition and biochemical approaches we demonstrated that instead of undergoing the ubiquitin-proteasome pathway, IKZF1 was degraded through apoptosis induced by kinase inhibition. This result may provide a new direction in developing therapeutic treatments for myeloma patients.

A lysine-to-arginine mutation on NEDD8 markedly reduces the activity of cullin RING E3 ligase through the impairment of neddylation cascades.

Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement of this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions.

Coupled local translation and degradation regulate growth cone collapse.

Local translation mediates axonal responses to Semaphorin3A (Sema3A) and other guidance cues. However, only a subset of the axonal proteome is locally synthesized, whereas most proteins are trafficked from the soma. The reason why only specific proteins are locally synthesized is unknown. Here we show that local protein synthesis and degradation are linked events in growth cones. We find that growth cones exhibit high levels of ubiquitination and that local signalling pathways trigger the ubiquitination and degradation of RhoA, a mediator of Sema3A-induced growth cone collapse. Inhibition of RhoA degradation is sufficient to remove the protein-synthesis requirement for Sema3A-induced growth cone collapse. In addition to RhoA, we find that locally translated proteins are the main targets of the ubiquitin-proteasome system in growth cones. Thus, local protein degradation is a major feature of growth cones and creates a requirement for local translation to replenish proteins needed to maintain growth cone responses.

Osteogenic effects of a potent Src-over-Abl-selective kinase inhibitor in the mouse.

Src-null mice have higher bone mass because of decreased bone resorption and increased bone formation, whereas Abl-null mice are osteopenic, because of decreased bone formation. Compound I, a potent inhibitor of Src in an isolated enzyme assay (IC(50) 0.55 nM) and a Src-dependent cell growth assay, with lower activity on equivalent Abl-based assays, potently, but biphasically, accelerated differentiation of human mesenchymal stem cells to an osteoblast phenotype (1-10 nM). Compound I (≥0.1 nM) also activated osteoblasts and induced bone formation in isolated neonatal mouse calvariae. Compound I required higher concentrations (100 nM) to inhibit differentiation and activity of osteoclasts. Transcriptional profiling (TxP) of calvaria treated with 1 µM compound I revealed down-regulation of osteoclastic genes and up-regulation of matrix genes and genes associated with the osteoblast phenotype, confirming compound I's dual effects on bone resorption and formation. In addition, calvarial TxP implicated calcitonin-related polypeptide, ß (ß-CGRP) as a potential mediator of compound I's osteogenic effect. In vivo, compound I (1 mg/kg s.c.) increased vertebral trabecular bone volume 21% (microcomputed tomography) in intact female mice. Increased trabecular volume was also detected histologically in a separate bone, the femur, particularly in the secondary spongiosa (100% increase), which underwent a 171% increase in bone formation rate, a 73% increase in mineralizing surface, and a 59% increase in mineral apposition rate. Similar effects were observed in ovariectomized mice with established osteopenia. We conclude that the Src inhibitor compound I is osteogenic, presumably because of its potent stimulation of osteoblast differentiation and activation, possibly mediated by ß-CGRP.

LPA induces osteoblast differentiation through interplay of two receptors: LPA1 and LPA4.

The bioactive phospholipid, lysophosphatidic acid (LPA), acting through at least five distinct receptors LPA1-LPA5, plays important roles in numerous biological processes. Here we report that LPA induces osteoblastic differentiation of human mesenchymal stem cells hMSC-TERT. We find that hMSC-TERT mostly express two LPA receptors, LPA1 and LPA4, and undergo osteoblastic differentiation in serum-containing medium. Inhibition of LPA1 with Ki16425 completely abrogates osteogenesis, indicating that this process is mediated by LPA in the serum through activation of LPA1. In contrast to LPA1, down-regulation of LPA4 expression with shRNA significantly increases osteogenesis, suggesting that this receptor normally exerts negative effects on differentiation. Mechanistically, we find that in hMSC-TERT, LPA induces a rise in both cAMP and Ca(2+). The rise in Ca(2+) is completely abolished by Ki16425, whereas LPA-mediated cAMP increase is not sensitive to Ki16425. To test if LPA signaling pathways controlling osteogenesis in vitro translate into animal physiology, we evaluated the bones of LPA4-deficient mice. Consistent with the ability of LPA4 to inhibit osteoblastic differentiation of stem cells, LPA4-deficient mice have increased trabecular bone volume, number, and thickness.

Hit to lead studies on (hetero)arylpyrimidines--agonists of the canonical Wnt-beta-catenin cellular messaging system.

A series of (hetero)arylpyrimidines agonists of the Wnt-beta-catenin cellular messaging system have been prepared. These compounds show activity in U2OS cells transfected with Wnt-3a, TCF-luciferase, Dkk-1 and tk-Renilla. Selected compounds show minimal GSK-3beta inhibition indicating that the Wnt-beta-catenin agonism activity most likely comes from interaction at Wnt-3a/Dkk-1. Two examples 1 and 25 show in vivo osteogenic activity in a mouse calvaria model. One example 1 is shown to activate non-phosphorylated beta-catenin formation in bone.

The effect of hydrogen peroxide solution on SO2 removal in the semidry flue gas desulfurization process.

The present study attempts to use hydrogen peroxide solution to humidify Ca(OH)(2) particles to enhance the absorption of SO(2) to achieve higher removal efficiency and to solve the valuable reuse of the reaction product in the semidry flue gas desulfurization (FGD) process. Experiments were carried out to examine the effect of various operating parameters including hydrogen peroxide solution concentration, Ca/S molar ratio and approach to adiabatic saturation temperature on SO(2) removal efficiency in a laboratory scale spray reactor. The product samples were analyzed to obtain semi-quantitative measures of mineralogical composition by X-ray diffraction (XRD) with reference intensity ratio (RIR) method and the morphology of the samples was examined by scanning electron microscope (SEM). Compared with spraying water to humidify Ca(OH)(2), SO(2) removal efficiency was improved significantly by spraying hydrogen peroxide solution of 1-3 wt.% to humidify Ca(OH)(2) because hydrogen peroxide solution enhanced the dissolution and absorption rate of SO(2). Moreover, XRD and SEM analyses show that the desulfurization products contain less amount of unreacted Ca(OH)(2) and more amount of stable calcium sulfate with increasing hydrogen peroxide solution concentration. Thus, the process mechanism of the enhanced absorption of SO(2) by spraying hydrogen peroxide solution to humidify Ca(OH)(2) was elucidated on the basis of the experimental results.

Identification of a PTH regulated gene selectively induced in vivo during PTH-mediated bone formation.

The biological activities of parathyroid hormone (PTH) on bone are quite complex as demonstrated by its catabolic and anabolic activities on the skeleton. Although there have been many reports describing genes that are regulated by PTH in osteoblast cells, the goal of this study was to utilize a well-established in vivo PTH anabolic treatment regimen to identify genes that mediate bone anabolic effects of PTH. We identified a gene we named PTH anabolic induced gene in bone (PAIGB) that has been reported as brain and acute leukemia cytoplasmic (BAALC). Therefore, using the latter nomenclature, we have discovered that BAALC is a PTH-regulated gene whose mRNA expression was selectively induced in rat tibiae nearly 100-fold (maximal) by a PTH 1-34 anabolic treatment regimen in a time-dependent manner. Although BAALC is broadly expressed, PTH did not regulate BAALC expression in other PTH receptor expressing tissues and we find that the regulation of BAALC protein by PTH in vivo is confined to mature osteoblasts. Further in vitro studies using rat UMR-106 osteoblastic cells show that PTH 1-34 rapidly induces BAALC mRNA expression maximally by 4 h while the protein was induced by 8 h. In addition to being regulated by PTH 1-34, BAALC expression can also be induced by other bone forming factors including PGE(2) and 1,25 dihydroxy vitamin D(3). We determined that BAALC is regulated by PTH predominantly through the cAMP/PKA pathway. Finally, we demonstrate in MC3T3-E1 osteoblastic cells that BAALC overexpression regulates markers of osteoblast differentiation, including downregulating alkaline phosphatase and osteocalcin expression while inducing osteopontin expression. We also demonstrate that these transcriptional responses mediated by BAALC are similar to the responses elicited by PTH 1-34. These data, showing BAALC overexpression can mimic the effect of PTH on markers of osteoblast differentiation, along with the observations that BAALC is induced selectively with a bone anabolic treatment regimen of PTH (not a catabolic treatment regimen), suggest that BAALC may be an important mediator of the PTH anabolic action on bone cell function.