Endogenous inhibins regulate steroidogenesis in mouse TM3 Leydig cells by altering SMAD2 signalling.

This study tested the hypothesis that inhibins act in an autocrine manner on Leydig cells using a pre-pubertal Leydig cell line, TM3, as a model of immature Leydig cells. The expression of Inha, Inhba, and Inhbb in TM3 cells was determined by RT-PCR and the production of the inhibin-alpha subunit was confirmed by western blot. Knockdown of Inha expression resulted in significant decreases in the expression of Leydig cell markers Cyp17a1, Cyp11a1, Nr5a1, and Insl3. Western blot showed that activin A, TGFß1 and TGFß2 activated SMAD2, and that knockdown of Inha expression in TM3 cells enhanced both activin A- and TGFß-induced SMAD2 activation. SB431542, a chemical inhibitor of the TGFß/activin type I receptors, blocked ligand-induced SMAD2 activation and the downregulation of Cyp17a1 expression. Our findings demonstrate that TGFßs and activin A negatively regulate steroidogenic gene expression in TM3 cells via ALK4/5 and SMAD2 and endogenous inhibins can counter this regulation.

Transcriptional responses of host peripheral blood cells to tuberculosis infection.

Host responses following exposure to Mycobacterium tuberculosis (TB) are complex and can significantly affect clinical outcome. These responses, which are largely mediated by complex immune mechanisms involving peripheral blood cells (PBCs) such as T-lymphocytes, NK cells and monocyte-derived macrophages, have not been fully characterized. We hypothesize that different clinical outcome following TB exposure will be uniquely reflected in host gene expression profiles, and expression profiling of PBCs can be used to discriminate between different TB infectious outcomes. In this study, microarray analysis was performed on PBCs from three TB groups (BCG-vaccinated, latent TB infection, and active TB infection) and a control healthy group. Supervised learning algorithms were used to identify signature genomic responses that differentiate among group samples. Gene Set Enrichment Analysis was used to determine sets of genes that were co-regulated. Multivariate permutation analysis (p < 0.01) gave 645 genes differentially expressed among the four groups, with both distinct and common patterns of gene expression observed for each group. A 127-probeset, representing 77 known genes, capable of accurately classifying samples into their respective groups was identified. In addition, 13 insulin-sensitive genes were found to be differentially regulated in all three TB infected groups, underscoring the functional association between insulin signaling pathway and TB infection.

Early transcriptional responses of HepG2-A16 liver cells to infection by Plasmodium falciparum sporozoites.

Invasion of hepatocytes by Plasmodium sporozoites deposited by Anopheles mosquitoes, and their subsequent transformation into infective merozoites is an obligatory step in the initiation of malaria. Interactions between the sporozoites and hepatocytes lead to a distinct, complex and coordinated cellular and systemic host response. Little is known about host liver cell response to sporozoite invasion, or whether it is primarily adaptive for the parasite, for the host, or for both. Our present study used gene expression profiling of human HepG2-A16 liver cells infected with Plasmodium falciparum sporozoites to understand the host early cellular events and factors influencing parasite infectivity and sporozoite development. Our results show that as early as 30 min following wild-type, non-irradiated sporozoite exposure, the expressions of at least 742 genes was selectively altered. These genes regulate diverse biological functions, such as immune processes, cell adhesion and communications, metabolism pathways, cell cycle regulation, and signal transduction. These functions reflect cellular events consistent with initial host cell defense responses, as well as alterations in host cells to sustain sporozoites growth and survival. Irradiated sporozoites gave very similar gene expression pattern changes, but direct comparative analysis between liver gene expression profiles caused by irradiated and non-irradiated sporozoites identified 29 genes, including glypican-3, that were specifically up-regulated only in irradiated sporozoites. Elucidating the role of this subset of genes may help identify the molecular basis for the irradiated sporozoites inability to develop intrahepatically, and their usefulness as an immunogen for developing protective immunity against pre-erythrocytic stage malaria.

Time course proteomic profiling of human myocardial infarction plasma samples: an approach to new biomarker discovery.

BACKGROUND: The aim of this study was to identify novel candidate biomarker proteins differentially expressed in the plasma of patients with early stage acute myocardial infarction (AMI) using SELDI-TOF-MS as a high throughput screening technology. METHODS: Ten individuals with recent acute ischemic-type chest pain (<12 h duration) and ST-segment elevation AMI (1STEMI) and after a second AMI (2STEMI) were selected. Blood samples were drawn at six times after STEMI diagnosis. The first stage (T0) was in Emergency Unit before receiving any medication, the second was just after primary angioplasty (T2), and the next four stages occurred at 12 h intervals after T0. Individuals (n=7) with similar risk factors for cardiovascular disease and normal ergometric test were selected as a control group (CG). Plasma proteomic profiling analysis was performed using the top-down (i.e. intact proteins) SELDI-TOF-MS, after processing in a Multiple Affinity Removal Spin Cartridge System (Agilent). RESULTS: Compared with the CG, the 1STEMI group exhibited 510 differentially expressed protein peaks in the first 48 h after the AMI (p<0.05). The 2STEMI group, had ~85% fewer differently expressed protein peaks than those without previous history of AMI (76, p<0.05). Among the 16 differentially-regulated protein peaks common to both STEMI cohorts (compared with the CG at T0), 6 peaks were persistently down-regulated at more than one time-stage, and also were inversed correlated with serum protein markers (cTnI, CK and CKMB) during 48 h-period after IAM. CONCLUSIONS: Proteomic analysis by SELDI-TOF-MS technology combined with bioinformatics tools demonstrated differential expression during a 48 h time course suggests a potential role of some of these proteins as biomarkers for the very early stages of AMI, as well as for monitoring early cardiac ischemic recovery.

Time course proteomic profiling of human myocardial infarction plasma samples: An approach to new biomarker discovery

Background: The aim of this study was to identify novel candidate biomarker proteins differentially expressedin the plasma of patients with early stage acute myocardial infarction (AMI) using SELDI-TOF-MS as a highthroughput screening technology.Methods: Ten individuals with recent acute ischemic-type chest pain (b12 h duration) and ST-segmentelevation AMI (1STEMI) and after a second AMI (2STEMI) were selected. Blood samples were drawn at sixtimes after STEMI diagnosis. The first stage (T0) was in Emergency Unit before receiving any medication, thesecond was just after primary angioplasty (T2), and the next four stages occurred at 12 h intervals after T0.Individuals (n=7) with similar risk factors for cardiovascular disease and normal ergometric test were selectedas a control group (CG). Plasma proteomic profiling analysis was performed using the top-down (i.e. intactproteins) SELDI-TOF-MS, after processing in a Multiple Affinity Removal Spin Cartridge System (Agilent).Results: Compared with the CG, the 1STEMI group exhibited 510 differentially expressed protein peaks in thefirst 48 h after the AMI (pb0.05). The 2STEMI group, had ~85% fewer differently expressed protein peaks thanthose without previous history of AMI (76, pb0.05). Among the 16 differentially-regulated protein peakscommon to both STEMI cohorts (compared with the CG at T0), 6 peaks were persistently down-regulated atmore than one time-stage, and also were inversed correlated with serum protein markers (cTnI, CK and CKMB)during 48 h-period after IAM.Conclusions: Proteomic analysis by SELDI-TOF-MS technology combinedwith bioinformatics tools demonstrateddifferential expression during a 48 h timecourse suggests a potential role of someof these proteins as biomarkersfor the very early stages of AMI, as well as for monitoring early cardiac ischemic recovery.

Reducing betaglycan expression by RNA interference (RNAi) attenuates inhibin bioactivity in LbetaT2 gonadotropes.

Betaglycan is an inhibin-binding protein co-receptor, the forced expression of which confers inhibin responsiveness on cells previously non-responsive to inhibin. The present study determines whether removal of betaglycan expression in otherwise inhibin-responsive cells will render the cells insensitive to inhibin. Small interfering RNAs (siRNAs) designed to the betaglycan gene were transfected into LbetaT2 gonadotrope cells to 'knock-down' betaglycan expression. To control for non-specific effects, siRNAs corresponding to an unrelated sequence (BF-1) were used. Two activin-responsive promoter constructs were used to assess inhibin bioactivity; an ovine FSHbeta promoter (oFSHbeta-lux), and a construct containing three copies of the activin-responsive sequence from the GnRHR promoter (3XpGRAS-PRL-lux). Activin stimulated the activity of both promoters 5-8-fold. Inhibin suppressed these activin-stimulated promoter activities by 52+/-11% and 51+/-7%, respectively. Similar inhibin suppression was also seen for cells co-transfected with the control BF-1 siRNAs. In contrast, inhibin's ability to suppress activin-stimulated activity was significantly reduced (33+/-3%, p<0.005 and 24+/-4%, p<0.045, respectively) in cells co-transfected with betaglycan siRNAs. These results demonstrated that endocrine effects of inhibin as a negative feedback controller of FSH production in gonadotropes are dependent on betaglycan expression.

Functional analysis of the human inhibin alpha subunit variant A257T and its potential role in premature ovarian failure.

BACKGROUND: A nucleotide substitution in the inhibin alpha subunit (INHA 769G>A, A257T) has been associated with premature ovarian failure (POF). We hypothesize this mutation causes a reduction in inhibin bioactivity, removing its suppression on the pituitary FSH secretion. The aim of this study is to establish if A257T inhibin has reduced bioactivity. METHODS: Mouse LbetaT2 pituitary gonadotrope, human granulosa (COV434) and human embryonic kidney (HEK293) cells were co-transfected with an activin-responsive reporter and increasing amounts of wild-type or variant A257T inhibin alpha subunit, and the degree of inhibin antagonism of activin signalling determined. RESULTS: A 5-fold inhibition was observed with wild-type inhibin alpha subunit overexpression (P < 0.001) (confirmed in HEK293 cells), while the A257T inhibin showed no inhibitory activity. In human ovarian COV434 transfected cells, while wild-type and A257T inhibin A had similar bioactivities, there was a significant reduction in the bioactivity of A257T inhibin B compared with wild-type inhibin B (P < 0.005). In all the three cell systems, overexpression of wild-type and A257T alpha subunit resulted in a 2- to 6-fold increase in secretion of dimeric inhibin indicating the reduced inhibin response was not due to a failure of dimerization. CONCLUSIONS: This study supports the hypothesis that the INHA 769G>A variant may increase susceptibility to POF with impaired inhibin B bioactivity and provides insight into the complex aetiology of POF.

Transforming growth factor-beta blocks inhibin binding to different target cell types in a context-dependent manner through dual mechanisms involving betaglycan.

Inhibin antagonizes activin and bone morphogenetic protein actions by sequestering their type II receptors in high-affinity complexes with betaglycan, a coreceptor that inhibin shares with TGF-beta. To clarify the nature and extent of interactions between inhibin and TGF-beta, we therefore examined 1) the mutual competition between these ligands for binding, 2) the regulation of endogenous betaglycan expression by inhibin and TGF-beta isoforms, and 3) the consequences of such betaglycan regulation for subsequent inhibin binding in mouse Leydig (TM3), Sertoli (TM4), adrenocortical cancer (AC), and gonadotroph (LbetaT2) cell lines, chosen to model cellular targets for local and endocrine actions of inhibin. Recognized inhibin, activin, and TGF-beta binding proteins and TGF-beta/activin signaling components were expressed by all four cell types, but AC and LbetaT2 cells notably lacked the type II receptor for TGF-beta, TbetaRII. Overnight treatment of TM3 and TM4 cells with TGF-beta1 suppressed the levels of betaglycan mRNA by 73 and 46% of control and subsequent [(125)I]inhibin A binding by 64 and 41% of control (IC(50) of 54 and 92 pm), respectively. TGF-beta2 acted similarly. TGF-beta pretreatments commensurately decreased the [(125)I]inhibin A affinity labeling of betaglycan on TM3 and TM4 cells. TGF-beta isoforms as direct competitors blocked up to 60% of specific inhibin A binding sites on TM3 and TM4 cells but with 9- to 17-fold lower potency than when acting indirectly via regulation of betaglycan. Only the competitive action of TGF-beta was observed with TbetaRII-deficient AC and LbetaT2 cells. Neither inhibin A nor inhibin B regulated betaglycan mRNA or competed for binding of [(125)I]TGF-beta1 or -beta2. Thus, inhibin binding to its target cell types is controlled by TGF-beta through dual mechanisms of antagonism, the operation of which vary with cell context and display different sensitivities to TGF-beta. In contrast, TGF-beta binding is relatively insensitive to the presence of either inhibin A or inhibin B.

Inhibins differentially antagonize activin and bone morphogenetic protein action in a mouse adrenocortical cell line.

Inhibin, a member of the TGF-beta superfamily, has been proposed to act as an inhibitor of activin and bone morphogenetic protein (BMP) by sequestering their type II receptors in nonsignaling complexes with betaglycan. This mechanism of inhibin action was tested in a mouse adrenocortical (AC) cell line by examining the effects of inhibins A and B on cytochrome P450 17alpha-hydroxylase 17,20-lyase (Cyp17) expression and 17alpha-hydroxylase activity, measured by progesterone 17alpha-hydroxylation, in the absence and presence of activin or BMP isoforms. Cyp17 mRNA endogenously expressed by AC cells was suppressed by activins A and B and BMP-2, -6, and -7, and each ligand accordingly inhibited 17alpha-hydroxyprogesterone production (IC(50) of 0.24, 0.27, 0.4, 0.51, and 2.2 nm, respectively). Neither inhibin A nor inhibin B alone affected Cyp17 expression or 17alpha-hydroxyprogesterone production. Both inhibin A and inhibin B blocked the inhibitory actions of activins A and B in AC cells, supporting the antiactivin model of inhibin action. Inhibin A provided more potent and effective antagonism of both activins than did inhibin B, and activin A was less subject to antagonism by either inhibin than was activin B. In contrast to the major antagonism of activin by both inhibins, only inhibin A antagonized the actions of BMP-2, BMP-6, and BMP-7, whereas inhibin B was ineffective against all tested BMP isoforms except BMP-7 at high concentrations. These results provide limited support for the anti-BMP model of inhibin action and reveal that, relative to inhibin A, inhibin B essentially behaves as a selective activin antagonist in AC cells. In conclusion, inhibins A and B differentially antagonize the actions of activins and BMPs to control adrenocortical C(19) steroid production.

Rodent adrenocortical cells display high affinity binding sites and proteins for inhibin A, and express components required for autocrine signalling by activins and bone morphogenetic proteins.

Inhibins are expressed in the adrenal cortex, but little is known of their binding or role in the adrenal. The aims of the present study were, first, to establish whether a mouse adrenocortical (AC) cell line expresses inhibins/activins and bone morphogenetic proteins (BMP), along with proteins required for inhibin to antagonise activin and BMP actions and, secondly, to characterise and compare inhibin binding sites and proteins in the rat adrenal gland and AC cells. AC cells were found to: (1) express mRNA for multiple BMPs (BMP-2, -3, -4, -6, -8a), growth/differentiation factors (GDF-1, -3, -5, -9), Lefty A and B, and the inhibin alpha, beta(A) and beta(B) subunits (2) secrete inhibin A and inhibin B and (3) express mRNA encoding the inhibin co-receptor, betaglycan, along with activin and BMP type I (ALK2-7) and type II (ActRII, ActRIIB, BMPRII) receptors, and binding proteins (follistatin, BAMBI, gremlin). When applied to sections of rat adrenal glands, [(125)I]inhibin A specifically bound to cells of the adrenal cortex, mainly in the zona reticularis. Scatchard analyses of in vitro [(125)I]inhibin A binding to dispersed rat adrenal cells and AC cells revealed sites of high affinity (K(d)(1) of 0.18 and 0.15 nM, respectively) and low affinity (K(d)(2) of 2.6 and 1.3 nM, respectively. Competition for [(125)I]inhibin A binding by activin A or B (30 nM) was negligible, whereas BMP-2, -6 and -7 competed for between 21 and 33% of specific inhibin A binding (IC(50) between 0.2 and 0.3 nM). Inhibin B crossreaction with inhibin A binding sites was < 8%. Multiple binding protein complexes (molecular weight ranging from 35 to > 220 kDa) were affinity labelled by [(125)I]inhibin A on both the primary rat adrenal and AC cells. The species of > 220 kDa were shown by immunoprecipitation to include betaglycan, the species of 105 kDa is consistent in size with type II receptors for activin/BMP, and that of 62 kDa co-migrates with the inhibin-follistatin complex. In summary, the results show that inhibin A binds selectively and with both high and low affinity to AC cells via multiple binding proteins, including a single betaglycan-like species. The results support the role of glycosylated betaglycan in the high affinity binding of inhibin A, but provide consistent evidence from two independent sources of adrenal cells that inhibin A interacts with several membrane proteins in addition to those currently understood to mediate the anti-activin/BMP actions of inhibin.

Labor-associated regulation of prostaglandin E and F synthesis and action in the ovine amnion and cervix.

OBJECTIVE: Prostaglandins (PGs) are key regulators of cervical dilatation and membrane breakdown at the onset of labor. PG synthase and receptor expression has been previously documented in uterine tissues; however, mechanisms governing the changes occurring in the cervix and amnion are less well established. The aim of the current study was to determine the level of expression of PG synthetic enzymes and receptors in these tissues in association with induced labor in sheep. METHODS: Labor was induced in sheep at 135 days of gestation by continuous fetal dexamethasone infusion. Amnion and cervical tissue was obtained before and after labor for measurement of mRNA encoding enzymes (cytosolic phospholipase A2 [cPLA2], PGH synthase-2 [PGHS-2], PGF synthase [PGFS], and PGE synthase [PGES]) and receptors (FP and EP1-4) by real-time polymerase chain reaction (PCR). RESULTS: cPLA2 expression increased significantly in cervical tissue at labor onset, whereas expression of the other enzymes measured did not change. There was a marked rise in EP3 expression in the cervix, but abundance of this receptor was lower than EP2 and FP expression, which did not change. The amnion exhibited a labor-associated decrease in PGHS-2, PGFS, and FP mRNA expression. CONCLUSION: The regulation of PG synthesis and action occurring in the amnion and cervix in association with labor appear to differ markedly between the two tissues, indicating tissue-specific roles for PGs. The data support a role for increased PG synthesis and action in the cervix and suggest a decrease in PG production and action in the amnion, in sharp contrast to the pattern reported in human amnion.

Prostaglandin e and f receptor expression and myometrial sensitivity at labor onset in the sheep.

Prostaglandins (PGs) play a pivotal role in the initiation and progression of term and preterm labor. Uterine activity is stimulated primarily by PGE(2) and PGF(2alpha) acting on prostaglandin E (EP) and prostaglandin F (FP) receptors, respectively. Activation of FP receptors strongly stimulates the myometrium, whereas stimulation of EP receptors may lead to contraction or relaxation, depending on the EP subtype (EP1-4) expression. Thus, the relative expression of FP and EP1-4 may determine the responsiveness to PGE(2) and PGF(2alpha). The aims of this study were to characterize the expression of EP1-4 and FP in intrauterine tissues and placentome, together with myometrial responsiveness to PG, following the onset of dexamethasone-induced preterm and spontaneous term labor. Receptor mRNA expression was measured using quantitative real-time polymerase chain reaction using species-specific primers. There was no increase in myometrial contractile receptor expression at labor onset, nor was there a change in sensitivity to PGE(2) and PGF(2alpha). This suggests expression of these receptors reaches maximal levels by late gestation in sheep. Placental tissue showed a marked increase in EP2 and EP3 receptor expression, the functions of which are unknown at this time. Consistent with previous reports, these results suggest that PG synthesis is the main factor in the regulation of uterine contractility at labor. This is the first study to simultaneously report PG E and F receptor expression in the key gestational tissues of the sheep using species-specific primers at induced-preterm and spontaneous labor onset.

Pituitary cell lines and their endocrine applications.

The pituitary gland is an important component of the endocrine system, and together with the hypothalamus, exerts considerable influence over the functions of other endocrine glands. The hypothalamus either positively or negatively regulates hormonal productions in the pituitary through its release of various trophic hormones which act on specific cell types in the pituitary to secrete a variety of pituitary hormones that are important for growth and development, metabolism, reproductive and nervous system functions. The pituitary is divided into three sections-the anterior lobe which constitute the majority of the pituitary mass and is composed primarily of five hormone-producing cell types (thyrotropes, lactotropes, corticotropes, somatotropes and gonadotropes) each secreting thyrotropin, prolactin, ACTH, growth hormone and gonadotropins (FSH and LH) respectively. There is also a sixth cell type in the anterior lobe-the non-endocrine, agranular, folliculostellate cells. The intermediate lobe produces melanocyte-stimulating hormone and endorphins, whereas the posterior lobe secretes anti-diuretic hormone (vasopressin) and oxytocin. Representative cell lines of all the six cell types of the anterior pituitary have been established and have provided valuable information on genealogy of the various cell lineages, endocrine feedback control of hormone synthesis and secretions, intrapituitary interactions between the various cell types, as well as the role of specific transcription factors that determine each differentiated cell phenotype. In this review, we will discuss the morphology and function of the cell types that make up the anterior pituitary, and the characteristics of the various functional anterior pituitary cell systems that have been established to be representative of each anterior pituitary cell lineage.

A novel serine protease of the mammalian HtrA family is up-regulated in mouse uterus coinciding with placentation.

This paper characterizes a novel gene, previously identified as uniquely regulated at implantation in mouse uterus. We cloned its full mRNA sequence encoding a serine protease possessing an IGF-binding domain and named it pregnancy-related serine protease (PRSP). PRSP is structurally similar to mammalian HtrA1 (56% amino acid similarity). Northern analysis revealed that the expression of PRSP mRNA was low before pregnancy, but it was increased at implantation and markedly up-regulated post-implantation. In-situ hybridization localized low levels of mRNA expression to the epithelium and stroma during very early pregnancy, but high expression to the decidual cells on day 8.5, primarily at the mesometrial pole where the placenta was forming. By day 10.5, PRSP mRNA was detected in the placenta. We also cloned an alternatively spliced PRSP mRNA that is expressed at a very low level. We located PRSP gene on chromosome 5 and established its intron/exon structure, which unambiguously explains how the two mRNA variants are produced through alternative splicing. Based on PRSP protein domain structure and its unique expression during pregnancy, we propose that PRSP plays an important role in the formation/function of the placenta.

Transforming growth factor-beta modulates inhibin A bioactivity in the LbetaT2 gonadotrope cell line by competing for binding to betaglycan.

Activin stimulates expression of GnRH receptor (GnRHR) and FSH beta-subunit in gonadotropes. Inhibin antagonizes activin actions on the gonadotropes, but its molecular mechanism of action remains poorly understood. It has been suggested that inhibin exerts its antagonistic effects by competing with activin for the binding of the activin receptor complex. Betaglycan has recently been identified as an inhibin-binding accessory protein in this process. Because both inhibin and TGFbeta bind betaglycan, we examined whether TGFbeta can modify inhibin's antagonism of activin-induced transcription in gonadotrope cells. Two activin-responsive reporter constructs were used, the first containing 5.5 kb of the ovine FSHbeta promoter (oFSHbetaluc), and the second containing three copies of the activin-responsive sequence of the GnRHR promoter (3XGRAS-PRL-lux). These constructs were transfected into the gonadotrope cell line LbetaT2. The oFSHbetaluc and 3XGRAS-PRL-lux activities stimulated by 0.5 nM activin A were decreased by up to 50% in a dose-dependent manner by inhibin A. TGFbeta(1) and TGFbeta(2) (0-4 nM), alone or in the presence of activin A, did not significantly affect the promoter elements. However, with increasing doses of TGFbeta(1) or TGFbeta(2), inhibin A antagonism of activin A activity was partly or completely reversed. Competition studies with radiolabeled inhibin A showed that TGFbeta(1) and TGFbeta(2) competed with [(125)I]inhibin for the binding to LbetaT2 cells (IC(50) = 280 pM and 72 pM, respectively). Immunoprecipitation studies of [(125)I]inhibin A cross-linked receptor complexes confirmed that TGFbeta(1) and TGFbeta(2) competed with inhibin A for the binding of betaglycan. These results suggest that TGFbeta competition with inhibin for binding to betaglycan interferes with inhibin's suppression of activin-induced FSHbeta and GnRHR promoters in LbetaT2 cells. We propose that under certain circumstances, TGFbeta may facilitate activin biological activity by hindering the access of inhibin to its coreceptor betaglycan.

Circulating levels of IGF-1 directly regulate bone growth and density.

IGF-1 is a growth-promoting polypeptide that is essential for normal growth and development. In serum, the majority of the IGFs exist in a 150-kDa complex including the IGF molecule, IGF binding protein 3 (IGFBP-3), and the acid labile subunit (ALS). This complex prolongs the half-life of serum IGFs and facilitates their endocrine actions. Liver IGF-1-deficient (LID) mice and ALS knockout (ALSKO) mice exhibited relatively normal growth and development, despite having 75% and 65% reductions in serum IGF-1 levels, respectively. Double gene disrupted mice were generated by crossing LID+ALSKO mice. These mice exhibited further reductions in serum IGF-1 levels and a significant reduction in linear growth. The proximal growth plates of the tibiae of LID+ALSKO mice were smaller in total height as well as in the height of the proliferative and hypertrophic zones of chondrocytes. There was also a 10% decrease in bone mineral density and a greater than 35% decrease in periosteal circumference and cortical thickness in these mice. IGF-1 treatment for 4 weeks restored the total height of the proximal growth plate of the tibia. Thus, the double gene disruption LID+ALSKO mouse model demonstrates that a threshold concentration of circulating IGF-1 is necessary for normal bone growth and suggests that IGF-1, IGFBP-3, and ALS play a prominent role in the pathophysiology of osteoporosis.