Synthesis of 17,20β,21‐trihydroxypregn‐4‐en‐3‐one by ovaries of reproductively mature Atlantic cod Gadus morhua

Atlantic cod Gadus morhua ovaries were incubated in vitro with tritiated 17‐hydroxypregn‐4‐ene‐3,20‐dione (17‐P) to determine whether 17,20β‐dihydroxypregn‐4‐en‐3‐one (17,20β‐P) or 17,20β, 21‐trihydroxypregn‐4‐en‐3‐one (17,20β,21‐P), or both, were more likely to be the steroid responsible for inducing oocyte final maturation (i.e. resumption of meiosis). Only 17,20β,21‐P was produced, in addition to 11‐deoxycortisol (17,21‐P), which is intermediate between 17‐P and 17,20β,21‐P. Also, the 5β‐reduced forms of 17‐P, 17,21‐P and 17,20β,21‐P were all found. Some sulphation of 21‐hydroxylated steroids was demonstrated. The ability of female G. morhua to make 17,20β,21‐P but not 17,20β‐P was confirmed by radioimmunoassay of plasma samples from spawning fish. Although small amounts of 17,20β‐P immunoreactivity were detected in a few plasma samples, this was shown, by thin‐layer chromatography, to be mostly due to cross‐reaction with other unidentified compounds. The evidence strongly suggests that 17,20β,21‐P is more likely than 17,20β‐P to be the maturation‐inducing steroid in G. morhua.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

Effect of defolliculation and 17α-hydroxy, 20β-dihydroprogesterone on cyclic AMP level in full-grown oocytes of the rainbow trout,Salmo gairdneri

The changes in cAMP were followed in trout oocytes incubated in vitro after defolliculation performed by either enzymatic or manual dissection. Both defolliculation methods induced a highly significant rise in oocyte cAMP level (4.5 times the basal level of control [follicle-enclosed oocytes], after 6 h). Treatment of defolliculated oocytes with 17α-hydroxy,20β-dihydroprogesterone (17α,20β-OH-P) (10^?6 M), which induced oocyte maturation (germinal vesicle breakdown [GVBD]) was able, first, to interrupt the increase of oocyte cAMP level promoted by defolliculation and then to lower this level significantly down to values that still remained higher than folliculated controls. Very low concentrations of 17α,20β-OH-P (1.38–55.6 10^?9 M), or physiological doses of testosterone (0.35 10^?6 M, in the range found in vivo before ovulation) were able to induce a similar decrease of oocyte cAMP level without inducing GVBD. Under the same experimental conditions estradiol (0.35 10^?6 M) exhibited no action. These results suggest that some factor(s) originating in the follicle (FIF), inhibit the oocytes' tendency to accumulate cAMP before the final surge of 17α,20β-OH-P. This factor might be a follicular steroid such as testosterone or nonmaturing concentrations of 17α,20β-OH-P. Moreover our data favour the hypothesis that the final surge of 17α,20β-OH-P could induce distinct intraoocyte mechanisms: the first induces an irreversible blockage of cAMP level before the inhibitory action of the FIF is suppressed by ovulation, and the second mechanism leads to GVBD.     

The effects of mass removal on the roach Rutilus rutilus (L.) stock of Lake Vesijärvi estimated with VPA within one season

The effects of mass removal on a roach stock was estimated with a virtual population analysis (VPA) within one season. High F/M ratio justifies the use of VPA in short intervals. The high ratio made it possible to obtain robust estimates because the terminal fishing mortality and the estimate of natural mortality had a negligible effect on the stock-size estimates. Trawling effectively removed almost all the roach from the trawling area. The instantaneous rate of fishing mortality reached values greater than 6.0 during the season in some age-groups. The estimated stock size was identical with weekly and monthly steps. The biomass of the stock was about 90 kg ha in May before the trawling was started. The results indicate that VPA is a convenient stock assessment method in biomanipulation projects especially if the population cannot avoid fishing by using refuges.     

Syntheses and anti‐tubercular activity of β‐substituted and α,β‐disubstituted peptidyl β‐aminoboronates and boronic acids

Tuberculosis is still affecting millions of people worldwide, and new resistant strains of Mycobacterium tuberculosis are being found. It is therefore necessary to find new compounds for treatment. In this paper, we report the synthesis and in vitro testing of peptidyl β‐aminoboronic acids and β‐aminoboronates with anti‐tubercular activity. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Reproductive behaviour and sexual production in the first‐generation hybrids of roach Rutilus rutilus L. × common bream Abramis brama L

The aim of this study was to further test the viability of the roach Rutilus rutilus × common bream Abramis brama hybrid in terms of reproductive behaviour and sexual production. Egg release, mating and aggressive acts in reproductive behaviour, as well as absolute fecundity and sperm density in milt for sexual production were examined in the first generation of these hybrids at their first sexual maturity. The F2 and backcrosses of hybrids were also studied. The results revealed that these hybrids expressed a normal and typical mating behaviour, producing viable gametes. Under experimental reproduction between hybrids (hybrid reproduction), the number of egg‐release acts (range, 21–66) was nearer (χ² test, P > 0.05) the number of mating acts (11–65). Moreover, hybrid males exhibited territorial and aggressive behaviours. However, in experimental reproduction of female and male hybrids mixed with parental males (mixed reproduction), the egg‐release act and the mating act were inhibited by the intense territorial and aggressive activities of the common bream male. Absolute fecundity values (median, <2.2 × 10³ eggs) and sperm density (<7 × 10⁹ spermatozoa ml^?1) of hybrids showed a greater decrease (U test, P < 0.05) than in parental species (median, >6.0 × 10³ eggs and >14 × 10⁹ spermatozoa ml^?1, respectively). Nevertheless, these hybrids were fertile. F2 and backcross generations were produced, although with a significantly lower viable hatching rate (FEP test, P < 0.05) in F2 individuals from the female and its corresponding hybrid male (<6%), indicating a very low chance of survival in rivers.     

Theaflavin‐3,3´‐digallate increases the antibacterial activity of β‐lactam antibiotics by inhibiting metallo‐β‐lactamase activity

Metallo‐β‐lactamases (MBLs) are some of the best known β‐lactamases produced by common Gram‐positive and Gram‐negative pathogens and are crucial factors in the rise of bacterial resistance against β‐lactam antibiotics. Although many types of β‐lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time‐kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand‐residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin‐3,3´‐digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time‐kill assays, we observed a synergistic effect of TFDG with β‐lactam antibiotics against methicillin‐resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with β‐lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of β‐lactam antibiotics against pathogens in vitro and in vivo.     

Sphingosine‐1‐phosphate (S1P) analog phytosphingosine‐1‐phosphate (P1P) improves the in vitro maturation efficiency of porcine oocytes via regulation of oxidative stress and apoptosis

Phytosphingosine‐1‐phosphate (P1P) is a signaling sphingolipid that regulates various physiological activities. However, little is known about the effect of P1P in the context of reproduction. Thus, we aimed to investigate the influence of P1P on oocyte maturation during porcine in vitro maturation (IVM). Here, we report the expression of S1PR1–3 among P1P receptors (S1PR1–4) in cumulus cells and oocytes. When P1P was administered at concentrations of 10, 50, 100, and 1,000 nM during IVM, the metaphase II rate was significantly increased in the 1,000 nM (1 μM) P1P treatment group. Maturation rate improvement by P1P supplementation was observed only in the presence of epidermal growth factor (EGF). Oocytes under the influence of P1P showed decreased intracellular reactive oxygen species levels but no significant differences in glutathione levels. In our molecular studies, P1P treatment upregulated gene expression involved in cumulus expansion (Has2 and EGF), antioxidant enzymes (SOD3 and Cat), and developmental competence (Oct4) while activating extracellular signal‐regulated kinase1/2 and Akt signaling. P1P treatment also influenced oocyte survival by shifting the ratio of Bcl‐2 to Bax while inactivating JNK signaling. We further demonstrated that oocytes matured with P1P displayed significantly higher developmental competence (cleavage and blastocyst [BL] formation rate) and greater BL quality (total cell number and the ratio of apoptotic cells) when activated via parthenogenetic activation (PA) and in vitro fertilization. Despite the low levels of endogenous P1P found in animals, exogenous P1P influenced animal reproduction, as shown by increased porcine oocyte maturation as well as preimplantation embryo development. This study and its findings are potentially relevant for both human and animal‐assisted reproduction.     

Melanopsin photoreception in the eye regulates light‐induced skin colour changes through the production of α‐MSH in the pituitary gland

How skin colour adjusts to circadian light/dark cycles is poorly understood. Melanopsin (Opn4) is expressed in melanophores, where in vitro studies suggest it regulates skin pigmentation through a ‘primary colour response’ in which light photosensitivity is translated directly into pigment movement. However, the entrainment of the circadian rhythm is regulated by a population of melanopsin‐expressing retinal ganglion cells (mRGCs) in the eye. Therefore, in vivo, melanopsin may trigger a ‘secondary colour response’ initiated in the eye and controlled by the neuro‐endocrine system. We analysed the expression of opn4m and opn4x and melanin aggregation induced by light (background adaptation) in Xenopus laevis embryos. While opn4m and opn4x are expressed at early developmental times, light‐induced pigment aggregation requires the eye to become functional. Pharmacological inhibition of melanopsin suggests a model whereby mRGC activation lightens skin pigmentation via a secondary response involving negative regulation of alpha‐melanocyte‐stimulating hormone (α‐MSH) secretion by the pituitary.     

The stress response neuropeptide CRF increases amyloid‐β production by regulating γ‐secretase activity

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid‐β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ‐secretase internalization. Co‐immunoprecipitation studies establish that γ‐secretase associates with CRFR1; this is mediated by β‐arrestin binding motifs. Additionally, CRFR1 and γ‐secretase co‐localize in lipid raft fractions, with increased γ‐secretase accumulation upon CRF treatment. CRF treatment also increases γ‐secretase activity in vitro, revealing a second, receptor‐independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ‐secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ‐secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ‐secretase.     

Immunomodulatory Activity of 3β,6β‐Dihydroxyolean‐12‐en‐27‐oic Acid in Tumor‐Bearing Mice

3β,6β‐Dihydroxyolean‐12‐en‐27‐oic acid ( 1 ) is a pentacyclic triterpenoid isolated from the rhizomes of Astilbe chinensis. To evaluate the in vivo antitumor potential and to elucidate its immunological mechanisms, effect of 1 on the growth of mouse‐transplantable tumors, and the immune response in naive and tumor‐bearing mice were investigated. The mice inoculated with mouse tumor cell lines were orally treated with 1 at the doses of 40, 60, and 80 mg/kg for 10 days. The effects of 1 on the growth of mouse‐transplantable S180 sarcoma and H22 hepatoma, splenocyte proliferation, cytotoxic T lymphocyte (CTL) activity, natural killer (NK) cell activity, and production of interleukin‐2 (IL‐2) from splenocytes in S180‐bearing mice were measured. Furthermore, the effect of 1 on 2,4‐dinitrofluorobenzene (DNFB)‐induced delayed‐type hypersensitivity (DTH) reactions and the sheep red blood cell (SRBC)‐induced antibody response in naive mice were also studied. Compound 1 could not only significantly inhibit the growth of mouse transplantable S180 sarcoma and H22 hepatoma, increase splenocytes proliferation, CTL and NK cell activity, and the level of IL‐2 secreted by splenocytes in tumor‐bearing mice, but also remarkably promote the DTH reaction and enhance anti‐SRBC antibody titers in naive mice. These results suggested that 1 could improve both cellular and humoral immune response, and could act as antitumor agent with immunomodulatory activity.     

The role of the maturation-inducing steroid, 17,20β-dihydroxypregn-4-en-3-one, in male fishes: a review

The major progestin in teleosts is not progesterone, as in tetrapods, but 17,20β-dihydroxypregn-4-en-3-one (17,20β-P) or, in certain species, 17,20β,21-trihydroxy-pregn-4-en-3-one (17,20β,21-P). Several functions for 17,20β-P and 17,20β,21-P have been proposed (and in some cases proved). These include induction of oocyte final maturation and spermiation (milt production), enhancement of sperm motility (by alteration of the pH and fluidity of the seminal fluid) and acting as a pheromone in male cyprinids. Another important function, initiation of meiosis (the first step in both spermatogenesis and oogenesis), has only very recently been proposed. This is a process that takes place at puberty in all fishes and once a year in repeat spawners. The present review critically examines the evidence to support the proposed functions of 17,20β-P in males, including listing of the evidence for the presence of 17,20β-P in the blood plasma of male fishes and discussion of why, in many species, it appears to be absent (or present at low and, in some cases, unvarying concentrations); consideration of the evidence, obtained mainly from in vitro studies, for this steroid being predominantly produced by the testis, for its production being under the control of luteinizing hormone (gonadotrophin II) and, at least in salmonids, for two cell types (Leydig cells and sperm cells) being involved in its synthesis; discussion of the factors involved in the regulation of the switch from androgen to 17,20β-P production that seems to occur in many species just at the time of spermiation; discussion of the effects of in vivo injection and application of 17,20β-P (and closely related compounds) in males; a listing of previously published evidence that supports the proposed new function of 17,20β-P as an initiator of meiosis; finally, discussion of the evidence for environmental endocrine disruption by progestins in fishes.     

Kappa opioids promote the proliferation of astrocytes via Gβγ and β‐arrestin 2‐dependent MAPK‐mediated pathways

GTP binding regulatory protein (G protein)‐coupled receptors can activate MAPK pathways via G protein‐dependent and ‐independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and β‐arrestin 2 pathways in kappa opioid receptor‐induced, extracellular signal‐regulated kinase 1/2 (ERK1/2)‐mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non‐nitrogenous agonist, C(2)‐methoxymethyl salvinorin B (MOM‐Sal‐B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gβγ subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of β‐arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM‐Sal‐B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM‐Sal‐B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gβγ subunits or β‐arrestin 2, suggesting that both G protein‐dependent and ‐independent ERK pathways are required for this outcome.     

Novel analogues of arginine vasopressin containing α‐2‐indanylglycine enantiomers in position 2

Continuing our efforts to obtain potent and selective analogues of AVP we synthesized and pharmacologically evaluated ten new compounds modified at position 2 with α‐2‐indanylglycine or its D ‐enantiomer (Igl or D ‐Igl, respectively). All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of these compounds to human OT receptor. The Igl² substitution resulted in a significant change of the pharmacological profile of the peptides. The new analogues were moderate or potent OT antagonists (pA₂ values ranging from 7.19 to 7.98) and practically did not interact with V_1a and V₂ receptors. It is worth emphasizing that these new peptides were exceptionally selective. On the other hand, the D ‐Igl² substituted counterparts turned out to be weak antagonists of the pressor response to AVP and displayed no antidiuretic activity. Some of the results were unexpected, e.g. dual activity in the rat uterotonic test in vitro: the D ‐Igl peptides showed a strong antioxytocic potency (pA₂ values ranging from 7.70 to 8.20) at low concentrations and full agonism at high concentrations. The results provided useful information about the SAR of AVP analogues. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The role of calcium in apoptosis induced by 7β‐hydroxycholesterol and cholesterol‐5β,6β‐epoxide

Oxysterols, such as 7β‐hydroxy‐cholesterol (7β‐OH) and cholesterol‐5β,6β‐epoxide (β‐epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca²⁺) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca²⁺ changes on apoptosis induced by 7β‐OH and β‐epoxide. Ca²⁺ responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura‐2. Over 15‐min exposure of differentiated U937 cells to 30 μM of 7β‐OH induced a slow but significant rise in fura‐2 ratio. The Ca²⁺ channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca²⁺. Moreover, dihydropyridine (DHP) binding sites identified with BODIPY‐FLX‐DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca²⁺ occurred through L‐type channels. However, following long‐term incubation with 7β‐OH, elevated levels of cytoplasmic Ca²⁺ were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca²⁺ may be an initial trigger of 7β‐OH–induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca²⁺ on apoptotic cell death appears to be less significant. In contrast, Ca²⁺ did not appear to be involved in β‐epoxide–induced apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:324–332, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20295     

β‐Bulges: Extensive structural analyses of β‐sheets irregularities

β‐Sheets are quite frequent in protein structures and are stabilized by regular main‐chain hydrogen bond patterns. Irregularities in β‐sheets, named β‐bulges, are distorted regions between two consecutive hydrogen bonds. They disrupt the classical alternation of side chain direction and can alter the directionality of β‐strands. They are implicated in protein‐protein interactions and are introduced to avoid β‐strand aggregation. Five different types of β‐bulges are defined. Previous studies on β‐bulges were performed on a limited number of protein structures or one specific family. These studies evoked a potential conservation during evolution. In this work, we analyze the β‐bulge distribution and conservation in terms of local backbone conformations and amino acid composition. Our dataset consists of 66 times more β‐bulges than the last systematic study (Chan et al. Protein Science 1993, 2:1574–1590). Novel amino acid preferences are underlined and local structure conformations are highlighted by the use of a structural alphabet. We observed that β‐bulges are preferably localized at the N‐ and C‐termini of β‐strands, but contrary to the earlier studies, no significant conservation of β‐bulges was observed among structural homologues. Displacement of β‐bulges along the sequence was also investigated by Molecular Dynamics simulations.     

Structures of single‐layer β‐sheet proteins evolved from β‐hairpin repeats

Free‐standing single‐layer β‐sheets are extremely rare in naturally occurring proteins, even though β‐sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single‐layer, anti‐parallel β‐sheet proteins, comprised of three or four twisted β‐hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of β‐sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent‐exposed tyrosine residues, was identified on the concave surface of the β‐sheet. These new modular single‐layer β‐sheet proteins may serve as a new model system for studying folding and design of β‐rich proteins.     

Role of Smad3, acting independently of transforming growth factor‐β, in the early induction of Wnt‐β‐catenin signaling by parathyroid hormone in mouse osteoblastic cells

Parathyroid hormone (PTH) exerts an anabolic action on bone but the mechanisms are incompletely understood. We showed previously that PTH interacts with the canonical Wnt‐β‐catenin signaling pathway via the transforming growth factor (TGF)‐β signaling molecule, Smad3, to modulate osteoblast differentiation and apoptosis. Here, we examined which actions of Smad3 are TGF‐β‐independent in stimulating the osteoblast phenotype and PTH‐induced Wnt‐β‐catenin signaling. For this, the TGF‐β receptor type 1 [activin receptor‐like kinase (ALK5)] inhibitor (SB431542), and a Smad3 mutant in which the site normally phosphorylated by ALK5 is mutated from SSVS to AAVA, was used. PTH induced total β‐catenin and reduced phosphorylated β‐catenin levels at 1, 6, and 24 h in mouse osteoblastic MC3T3‐E1 cells. Transient transfection of Smad3AAVA inhibited the PTH induction of total β‐catenin and reduction of phosphorylated β‐catenin levels at 6 and 24 h, but not at 1 h, indicating that the early effects occur independently of TGF‐β receptor signaling. On the other hand, MC3T3‐E1 cell clones in which Smad3AAVA was stably expressed demonstrated elevated β‐catenin levels, although alkaline phosphatase (ALP) activity and mineralization were unaltered. In contrast, MC3T3‐E1 cell clones in which wild‐type Smad3 was stably expressed exhibited increased ALP activity and mineralization that were decreased by the ALK5 inhibitor, SB431542, although the β‐catenin levels induced in these cells were not modulated. In conclusion, the present study indicates that PTH induces osteoblast β‐catenin levels via Smad3 independently of, and dependently on, TGF‐β in the early and later induction phases, respectively. J. Cell. Biochem. 108: 285–294, 2009. © 2009 Wiley‐Liss, Inc.