IGFBP-5 enhances epithelial cell adhesion and protects epithelial cells from TGFß1-induced mesenchymal invasion.

TGFß1 is a major fibrotic factor and its actions involve induction of epithelial cell death, together with the stimulation and transdifferentiation of fibroblasts into collagen- and fibronectin-secreting myofibroblasts. These actions of TGFß1 are also consistent with a pro-metastatic role, by aiding epithelial cell escape through mesenchymal tissues. Recently IGFBP-5 has been described as a pro-fibrotic (pro-metastatic?) agent and the aim of this study was to compare and contrast the actions of IGFBP-5 with TGFß1. We used NMuMG cells and cloned stable epithelial and mesenchymal lines from the parent cells. TGFß1 induced apoptosis and/or EMT in the epithelial cells, whereas it enhanced mesenchymal cell survival and migration. IGFBP-5, in contrast, enhanced both cell-cell and cell-ECM adhesion and also improved wound closure in epithelial cells whereas, in mesenchymal cells, IGFBP-5 decreased adhesion and migration. Furthermore, IGFBP-5 was able to antagonise the actions of TGFß1. In a co-culture model simulating epithelial-mesenchymal boundaries, IGFBP-5 was able to antagonise the disruptive transgressions induced by TGFß1. Overall, these findings suggest that IGFBP-5 is important in maintaining epithelial-mesenchymal boundaries and thus may limit metastasis and fibrosis by inducing an orderly repair mechanism, very distinct from the fibrotic disruption induced by TGFß1. A role for IGFBP-5 in the inhibition of metastasis is supported by immunohistochemical studies of breast cancer microarrays, where we show that elevated IGFBP-5 expression is associated with increased disease-free survival.

Relative Roles of TGF-ß and IGFBP-5 in Idiopathic Pulmonary Fibrosis.

Although most evident in the skin, the process of scarring, or fibrosis, occurs in all major organs because of impaired epithelial self-renewal. No current therapy exists for Idiopathic pulmonary fibrosis. The major profibrotic factor is TGF-ß1 and developing inhibitors is an area of active research. Recently, IGFBP-5 has also been identified as a profibrotic factor, and studies suggest that, while both TGF-ß1 and IGFBP-5 activate mesenchymal cells to increase collagen and fibronectin production, their effects on epithelial cells are distinct. TGF-ß1 induces cell death and/or EMT in the epithelial cells, exacerbating the disruption of tissue architecture. In contrast, IGFBP-5 induces epithelial cell spreading over collagen or fibronectin matrices, increases secretion of laminin, the epithelial basement membrane, and enhances the survival of epithelial cells in nutrient-poor conditions, as exists in scar tissue. Thus, IGFBP-5 may enhance repair and may be an important target for antifibrotic therapies.

Role of insulin-like growth factor binding proteins in mammary gland development.

Insulin-like growth factors (IGFs) play an important role in mammary gland development and their effects are, in turn, influenced by a family of 6 IGF-binding proteins (IGFBPs). The IGFBPs are expressed in time- and tissue-specific fashion during the periods of rapid growth and involution of the mammary gland. The precise roles of these proteins in vivo have, however, been difficult to determine. This review examines the indirect evidence (evolution, chromosomal location and roles in lower life-forms) the evidence from in vitro studies and the attempts to examine their roles in vivo, using IGFBP-deficient and over-expression models. Evidence exists for a role of the IGFBPs in inhibition of the survival effects of IGFs as well as in IGF-enhancing effects from in vitro studies. The location of the IGFBPs, often associated with the extracellular matrix, suggests roles as a reservoir of IGFs or as a potential barrier, restricting access of IGFs to distinct cellular compartments. We also discuss the relative importance of IGF-dependent versus IGF-independent effects. IGF-independent effects include nuclear localization, activation of proteases and interaction with a variety of extracellular matrix and cell surface proteins. Finally, we examine the increasing evidence for the IGFBPs to be considered as part of a larger family of extracellular matrix proteins involved in morphogenesis and tissue re-modeling.

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland.

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue remodeling processes. The ability to separate these properties, using mutant IGFBPs, is currently under investigation.

Hormonal control of plasmin and tissue-type plasminogen activator activity in rat milk during involution of the mammary gland.

We have proposed that growth hormone (GH) and prolactin (PRL) interact to suppress apoptosis in the mammary gland. GH increases insulin-like growth factor-I (IGF-I) synthesis whereas PRL suppresses the production of insulin-like growth factor-binding protein-5 (IGFBP-5) in the epithelial cells, which would otherwise inhibit IGF-mediated cell survival. IGFBP-5 was present in milk from involuting glands at high concentrations (approximately 60 microg/ml) and had a high affinity (8.03 x 10(-10) M) for IGF-I, suggesting an inhibitory effect of IGFBP-5 in the mammary gland. IGFBP-5 was present in the micellar fraction of milk and binds specifically to alpha(s2)-casein. Since alpha(s2)-casein also binds plasminogen and tissue-type plasminogen activator (t-PA), resulting in the conversion of plasminogen to plasmin, and since IGFBP-5 binds to plasminogen activator inhibitor-1 (PAI-1), we investigated whether apoptosis and extracellular matrix (ECM) degradation might be coordinately controlled by GH and PRL possibly acting through IGFBP-5. Litters were removed from lactating rats to initiate involution. Plasminogen activation and t-PA activity were both increased dramatically after 48 h and GH and PRL suppressed this response. By contrast, 17beta-oestradiol, progesterone or corticosterone did not influence either process. An antiserum to IGF-I, which blocked systemic IGF-I effects, failed to inhibit the activation of plasminogen or the increase in t-PA, suggesting that paracrine effects of IGF-I may be more important. Teat-sealing, which led to the accumulation of milk without hormonal changes, also led to increases in plasminogen activation and t-PA activity, suggesting that locally produced factors (of which IGFBP-5 is one) are important in controlling ECM remodelling. We propose that GH and PRL inhibit apoptosis and ECM remodelling by a process that involves the control of IGF-I and PAI-1 availability by IGFBP-5, thus allowing these processes to be tightly coordinated.

Insulin-like growth factor binding protein-5 (IGFBP-5) potentially regulates programmed cell death and plasminogen activation in the mammary gland.

This study aims to investigate the mechanism by which prolactin and GH interact to maintain mammary epithelial cell function in the rat. IGF-I is an important survival factor for the mammary gland and we have demonstrated that the effects of GH and prolactin involve IGF-I. GH acts by increasing IGF-I whilst prolactin acts by inhibiting the expression of IGFBP-5 from the mammary epithelium. During mammary involution, when serum prolactin levels decline, IGFBP-5 expression is dramatically upregulated and it binds with high affinity to IGF-I preventing IGF-I interaction with the IGF-receptor and thus leading to epithelial cell apoptosis. We have identified a specific interaction of IGFBP-5 with alpha s2-casein. This milk protein has also been shown to bind plasminogen and its activator tissue-type plasminogen activator (tPA) leading to enhanced conversion of plasminogen to plasmin. Plasmin is an important initiator of re-modelling of the extracellular matrix during mammary involution. A potential interaction between the cell death and extracellular matrix remodelling is evident from the observation that IGFBP-5 binds to plasminogen activator inhibitor-I (PAI-1). We thus hypothesized that IGFBP-5 could activate cell death by sequestration of IGF-I and activate plasminogen cleavage by sequestering PAI-1. In support of this hypothesis we have shown that both prolactin and GH inhibit tPA activity and plasminogen activation in the involuting mammary gland. Our results suggest that GH and prolactin inhibit cell death and ECM remodelling via the IGF-axis and also indicate a novel role for the milk protein alpha s2-casein in this process. We have now established lines of transgenic mice expressing IGFBP-5 on the beta-lactoglobulin promoter to explore its function in greater detail.

The role of Stat3 in apoptosis and mammary gland involution. Conditional deletion of Stat3.

STATs (signal transducer and activator of transcription) are a family of latent transcription factors which are activated in response to a variety of cytokines and growth factors. This family of signalling molecules have been implicated in growth, differentiation, survival and apoptosis. In this article, we will review work which highlights the role of individual STAT factors in mammary gland and demonstrate the value of genetically modified mice in defining the function of STAT3. Involution of the mouse mammary gland is characterised by extensive apoptosis of the epithelial cells and the activation of STAT3. STATs 3 and 5 have reciprocal patterns of activation throughout a mammary developmental cycle suggesting that STAT5 may be a survival factor and STAT3 a death factor for differentiated mammary epithelium. To clarify the role of STAT3 in mammary epithelial apoptosis, we have generated a conditional knockout using the lox/Cre recombination system. Mammary glands from crosses of transgenic mice expressing Cre recombinase under the control of the beta-lactoglobulin milk protein gene promoter with mice harbouring one floxed STAT3 allele and one null STAT3 allele, showed a decrease in epithelial apoptosis and a dramatic delay of the involution process upon forced weaning. This was accompanied by precocious activation of STAT1 and increases in p53 and p21 levels--these may act as a compensatory mechanism for initiating the eventual involution which occurs in STAT3 null mammary glands. This demonstrates for the first time the importance of STAT factors in signalling the initiation of physiological apoptosis in vivo and highlights the utility of the lox/Cre system for addressing the function of genes, which have an embryonic lethal phenotype, specifically in mammary gland.

Insulin-like growth factor binding proteins: IGF-dependent and -independent effects in the mammary gland.

The insulin-like growth factor binding proteins (IGFBPs) are a family of proteins which bind to the IGFs with high affinity. Their expression within the mammary gland is species specific; it has thus been difficult to determine the biological roles of these binding proteins during lactation. In this article we propose a role for IGFBP-5 in the mammary gland involving the initiation of apoptosis induced by sequestration of IGF-1, an important survival factor for the mammary gland. We have shown that this binding protein retains its high affinity for IGF-1 and that it is present in extremely high concentrations compared with the growth factor. These observations make it likely that IGFBP-5 is capable of preventing interaction of IGF-1 with its receptor on the epithelial cells synthesizing milk. We have also demonstrated that IGFBP-5 interacts with alpha(s2)-casein and that this interaction implicates it in the regulation of plasminogen activation in the mammary gland. The generation of plasmin is a key initiating event in the remodeling of the extracellular matrix during mammary involution. As such, IGFBP-5 may play a key role in coordinating cell death and tissue remodeling processes. Many of the molecules involved in embryological development are also expressed in the developing and involuting mammary gland. We believe that our studies may offer mechanistic explanations for apoptotic events in a wide variety of tissues. We have recently shown that IGFBP-5 is apoptotic in the chick embryonic limb bud, adding further support to our belief that IGFBP-5 serves this function in the mammary gland. We hope to be able to explore the role of this binding protein in the mammary gland with a transgenic mouse model expressing IGFBP-5 on the beta-lactoglobulin promoter.

A novel role for IRF-1 as a suppressor of apoptosis.

The tumour suppressor IRF-1 is a transcription factor involved in the induction of apoptosis in several in vitro systems. Post-lactational involution of the mammary gland is characterized by extensive apoptosis of the epithelial cells. We have previously shown that signal transducer and activator of transcription (Stat) 3 drives apoptosis and involution in the mouse mammary gland. Since one of the downstream targets of the Stat signalling pathway is IRF-1, we have used IRF-1 knockout mice to address the potential role of this transcription factor in involution. Surprisingly, in the absence of IRF-1 significantly higher numbers of apoptotic cells were found in involuting glands at 48 h compared to control glands. In addition, the alveolar structure in IRF-1 null mammary glands had collapsed whereas in control glands the alveoli remained intact and distended. However, by 72 h control and null glands were morphologically similar suggesting that IRF-1 suppresses apoptosis only during the early, reversible, stage of involution. This suggests a survival role for IRF-1 in mammary epithelia and demonstrates a novel role for IRF-1 in vivo--suppression of premature epithelial apoptosis during mammary gland involution.

Suppression of epithelial apoptosis and delayed mammary gland involution in mice with a conditional knockout of Stat3.

Mammary gland involution is characterized by extensive apoptosis of the epithelial cells. At the onset of involution, Stat3 is specifically activated. To address the function of this signaling molecule in mammary epithelial apoptosis, we have generated a conditional knockout of Stat3 using the Cre-lox recombination system. Following weaning, a decrease in apoptosis and a dramatic delay of involution occurred in Stat3 null mammary tissue. Involution is normally associated with a significant increase in IGFBP-5 levels. This was observed in control glands, but not in the absence of Stat3. IGFBP-5 has been suggested to induce apoptosis by sequestering IGF-1 to casein micelles, thereby inhibiting its survival function. Our findings suggest that IGFBP-5 is a direct or indirect target for Stat3 and its upregulation is essential to normal involution. No marked differences were seen in the regulation of Stat5, Bcl-x(L), or Bax in the absence of Stat3. Precocious activation of Stat1 and increases in levels of p53 and p21 occurred and may act as compensatory mechanisms for the eventual initiation of involution observed in Stat3 null mammary glands. This is the first demonstration of the importance of a Stat factor in signaling the initiation of physiological apoptosis in vivo.

Influence of microenvironment on mammary epithelial cell survival in primary culture.

Mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix form multicellular structures termed mammospheres, in which cells and matrix become arranged around a central luminal space. In the presence of lactogenic hormones, cells within mammospheres become polarized, form tight intercellular junctions, and secrete milk proteins vectorially into the luminal space. This study examined the mechanism of lumen formation. Histological examination of developing mammospheres showed that cavitation was associated spatially and temporally with the appearance of fragmented nuclear material in apoptotic bodies, and with the presence of cells positively labeled by terminal deoxynucleotide transferase-mediated deoxyuridine nick end-labeling (TUNEL). Analysis of [(32)P]-deoxynucleotide end-labeled genomic DNA by electrophoresis and autoradiography showed DNA laddering indicative of apoptosis. A transient increase in laddering coincided with both lumen formation and the presence of TUNEL-positive cells. Lumen formation, DNA laddering, and detection of TUNEL-positive cells were all accelerated when matrix composition was altered. They were also impaired coordinately when caspase inhibitor was present during the first two days of culture. Therefore, lumen formation in mammosphere cultures is due to selective apoptosis of centrally located cells. Mammosphere cavitation was accompanied by redistribution of matrix constituents to the mammosphere periphery. Western blotting and Western ligand blotting of culture medium showed that lumen formation was also associated with a transient increase in insulin-like growth factor binding protein-5 (IGFBP5), a factor implicated in mammary apoptosis in vivo. We propose that epithelial cell survival during mammosphere development is induced selectively through stabilization by basement membrane constituents, which may act directly on the epithelial cell or confer protection against autocrine apoptotic factors.

Amino acids within the extracellular matrix (ECM) binding region (201-218) of rat insulin-like growth factor binding protein (IGFBP)-5 are important determinants in binding IGF-I.

The highly conserved N-and C-terminal domains of IGFBPs are believed to participate in IGF binding, but only recently have some of the critical residues in the IGFBP sequence involved in ligand binding been identified. Here we describe two highly conserved amino acids in the C-terminal domain of rat IGFBP-5 that are involved in binding IGF-I. Site-directed mutagenesis was used to produce two mutants, G203K and Q209A, of rIGFBP-5. Relative to wild-type rIGFBP-5, an 8-fold reduction in affinity for human IGF-I was found for recombinant G203K protein in both IGF-I ligand blots and solution phase ligand binding assays, and a 7-and 6-fold reduction for Q209A respectively. This shows that Gly203 and Gln209 in IGFBP-5 are important determinants in binding IGF-I, and due to their complete conservation in all IGFBP sequences, we suggest that they are likely to be involved in binding IGF-I in all six binding proteins. In addition, these two non-basic residues lie within the ECM binding region (201-218) of IGFBP-5, demonstrating that the C-terminus contains partially overlapping IGF-I and ECM binding sites. We therefore propose that heparin binding to basic amino acids in IGFBP-5 between 201-218 may physically occlude subsequent interaction between IGF-I and Gly203/Gln209, and that this may explain previous work of others showing reduced affinity of ECM bound IGFBP-5 for IGF-I.

Hormonal control of insulin-like growth factor-binding protein-5 production in the involuting mammary gland of the rat.

We have demonstrated a 50-fold increase in the concentration of insulin-like growth factor-binding protein-5 (IGFBP-5) in milk after 2 days of mammary involution induced by removal of the suckling young. IGFBP-5 was identified by its immunoreactivity with an antiserum to IGFBP-5 and was shown by in situ hybridization to be synthesized by the secretory epithelial cells undergoing apoptosis. Smaller increases in IGFBP-2 and -4 messenger RNAs (mRNAs) were also evident, but neither protein could be detected on Western ligand blots of milk. Preliminary evidence failed to detect mRNAs for IGFBP-1, -3, or -6. The large increase in IGFBP-5 concentrations in milk from involuting mammary glands was inhibited by 90% if the dams received concurrent PRL injections for 2 days, but was unaffected by GH, progesterone, corticosterone, or an antiserum to insulin-like growth factor I (IGF-I). In lactating rats allowed to continue nursing their young, 17beta-estradiol failed to affect IGFBP-5 concentrations, whereas in animals that had half the teats sealed to prevent milk removal, IGFBP-5 concentrations increased 5- to 10-fold in the sealed gland compared with those in the contralateral gland where milk removal continued. The changes in IGFBP-5 concentrations in milk were accompanied by similar changes in steady state mRNA levels of IGFBP-5 in mammary tissue. We have previously shown that PRL inhibits apoptosis and involution of the mammary gland, whereas teat sealing has the opposite effect. We, therefore, propose that IGFBP-5 serves to inhibit IGF-I-mediated cell survival, but that it is normally suppressed by PRL and milk removal. Although IGFBP-5, when bound to extracellular matrix, augments the action of IGF, we believe that in the involuting mammary gland IGFBP-5 inhibits IGF action by interacting with casein micelles, which contain calcium phosphate nanoclusters, thereby preventing IGF interaction with IGF receptors. This is analogous to the interaction of IGFBP-5 with hydroxyapatite, which serves to sequester IGFs in bone. IGFBP-5 may, in fact, play a central role in inducing apoptosis, as it is also up-regulated in involuting prostate and thyroid glands as well as in atretic ovarian follicles.

The role of prolactin and growth hormone in the regulation of casein gene expression and mammary cell survival: relationships to milk synthesis and secretion.

We have compared involution of the rat mammary gland, induced by litter removal, where milk accumulation occurs, with involution induced in the presence of the suckling young by combined PRL and GH deficiency. Both treatments induced involutionary processes involving apoptosis, as judged by DNA ladders and resulted in significant decreases in the DNA content of the gland. Surprisingly, the effects of hormone deprivation on protein output in milk were principally explained by the loss of secretory cells, as there were only modest decreases in casein messenger RNA (mRNA) expression and protein synthesis rates per U DNA in vitro. The association of casein mRNA with the polysome fraction was also unaffected by hormone deprivation, whereas involution induced by litter removal resulted in much greater decreases in steady state levels of casein mRNA and an increased association of the mRNAs with the monosome fraction. In PRL- and GH-deficient rats, PRL treatment could prevent all of these effects, GH was partially effective, whereas putative mediators of GH action, insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding protein-3, were ineffective. This lack of effect of IGFs may be due to an inhibitory IGFBP, which we demonstrate to be present in increased amounts in the involuting mammary gland.

Effects of growth hormone and ovariectomy on performance, serum hormones, insulin-like growth factor-binding proteins, and muscle fiber properties of prepubertal Friesian heifers.

Sixteen prepubertal Friesian heifers were used to examine the effect of bovine growth hormone (GH) and ovariectomy (OVX) at 2.5 mo of age (2 x 2 factorial design) on growth, carcass quality, and fiber types, capillarization, and metabolic potentials of the longissimus muscle, and serum concentrations of estradiol-17beta (E2beta), insulin, GH, IGF-I, and IGF-binding proteins (IGFBP). Treatment with GH (15 mg/d) started at 147 +/- 3 kg BW and lasted for 15 wk. Heifers were fed a mixed roughage-based diet. Growth hormone increased ADG (P < .001), improved gain:feed (P < .007), and had a small but positive influence on lean accretion. Growth hormone reduced fat thickness (P < .009), carcass fat trim (P < .009) and i.m. fat (P < .09). Ovariectomy did not affect performance but increased dressing percentage (P < .03), full rib weight (P < .003), and fat thickness (P < .04). Ovariectomy reduced E2beta (P < .001) and insulin (P < .02), and increased the 32-kDa IGFBP (IGFBP-2) (P < .09). Growth hormone treatment increased GH, IGF-I, the 28-kDa IGFBP, and the 40- to 43-kDa IGFBP (IGFBP-3) (P < .004 or P < .001). Neither GH nor ovariectomy affected the proportion and relative area of the individual muscle fiber types, but GH tended to increase type I fiber area (P < .10). Number of capillaries per fiber increased in OVX GH-treated heifers (GH x OVX interaction, P < .02). Activities of citrate synthetase were higher in GH-treated (P < .05) and OVX (P < .02) heifers, indicating increased oxidative capacity of the longissimus muscle. The effects of GH on performance and carcass fattening were in accordance with the observed hormonal changes. When slaughter occurs before puberty, ovariectomy has no effect on performance, only few effects on carcass quality, and small effects on hormone concentrations.

Production of insulin-like growth factor binding protein (IGFBP), IGFBP-3 protease, and expression of IGF-I receptors by cells of the sheep immune system.

Single-cell suspensions of sheep thymus cells were cultured in serum-free medium with or without polyclonal activators (phytohaemagglutinin or concanavalin A) and the resultant conditioned medium was assayed for insulin-like growth factor binding protein (IGFBP) activity by binding of [125I]IGF-I, using charcoal to separate free from bound. All cultures produced IGFBP but mitogen stimulation significantly increased IGFBP concentrations, indicating production by lymphoid cells. Conditioned medium also degraded recombinant human [125I]IGFBP-3, suggesting IGFBP-3 protease production within the thymus. This degradation was inhibited by several protease inhibitors (phenylmethylsulphonyl fluoride, aprotinin, N-alpha-p-tosyl-L-lysine chloromethyl ketone), suggesting the presence of a serine protease. Cell surface [125I]IGF-I binding was demonstrated on cells from thymus, mesenteric lymph node, peripheral blood mononuclear cells and platelets. The [125I]IGF-I binding to platelets could be inhibited by unlabelled peptides, with relative potencies IGF-I > IGF-II >> insulin. Scatchard analysis of IGF-I competitive binding revealed a Kd of 266 pmol/l and approximately 40 receptor sites per cell. The high-affinity binding of IGF-I and competition by insulin suggested that the [125I]IGF-I binding was to an IGF-I receptor rather than to a membrane-associated IGFBP, to which insulin does not bind. These data provide further support for the role of the IGF-IGFBP axis in the immune system, particularly in relation to the thymus.

Does an IGF-binding protein (IGFBP) present in involuting rat mammary gland regulate apoptosis?

We examined the effects of GH and prolactin deficiency upon milk production, apoptosis and IGFBP production by the mammary gland. GH deficiency produced a 15% reduction in milk yield, prolactin a 50% reduction and combined prolactin- and GH-deficiency an 85% reduction in milk production. Litter removal led to complete inhibition of milk synthesis within 24 h owing in large part to milk accumulation. GH- and prolactin-deficiency also led to significant loss of mammary cells within 48 h and this was owing at least in part to apoptosis as judged by the appearance of characteristic DNA ladders. Prolactin replacement therapy could prevent all of these changes whilst GH was partially effective. The effects of GH are believed to be mediated via IGF-I, however, we were unable to mimic the effects of GH with IGF-I, IGF-II or a combination of IGF-I, IGF-II and IGFBP-3. We hypothesized that the cell-survival effects of exogenous IGFs might be blocked by an inhibitory IGFBP produced by the gland. Indeed the involuting mammary gland produces large concentrations of an IGFBP which Northern blotting identified as IGFBP-5. There was also a small increase in IGFBP-4 mRNA expression. Both appear to be produced by the secretory epithelial cells as judged by in situ hybridization. Preliminary studies using mouse "mammosphere" cultures suggest that they will be useful for investigating a potential causal relationship between IGFBP-5 synthesis and apoptosis.

Effects of a polyclonal antiserum to rat growth hormone on circulating insulin-like growth factor (IGF)-I and IGF-binding protein concentrations and the growth of muscle and bone.

A polyclonal antiserum to rat GH (anti-rGH) injected into rats for 3 or 8 weeks markedly reduced the weight, total protein and RNA content of muscles of the hind limb. These effects were prevented when bovine GH (bGH) was administered simultaneously. In a second experiment, the effects of 8 weeks of treatment with anti-rGH on the growth of the whole body, muscle and bone were investigated. Body weights of rats were decreased by 58% by treatment with anti-rGH; muscle weights were reduced by slightly more than the decrease in body weight (by 64%, 65% and 61% respectively for plantaris, soleus and gastrocnemius). The weight of the tibia was decreased by 54%, its length was decreased by 23%, cortical width and overall width were reduced by 26% and 18% respectively, suggesting a possible role for GH in osteoclastic activity. Serum total insulin-like growth factor-I (IGF-I) concentrations were decreased by 80-90% in both experiments by anti-rGH; these changes were prevented in the first experiment by concurrent treatment with anti-rGH and bGH. The serum IGF-binding protein-3 (IGFBP-3) concentration was also decreased by anti-rGH in experiment 1 (by 86%); the response of the 28-32 kDa IGFBPs was smaller (-35%), and was restored to control values by simultaneous injection of bGH. Western immunoblotting using an antiserum to IGFBP-2 showed that there was a marked decrease from neonatal to adult stages which was independent of anti-rGH treatment. This clearly demonstrated a dissociation of the reciprocal relationship supposed to exist between IGFBPs-2 and -3. The 24 kDa IGFBP-4 was unaffected by anti-rGH but replacement therapy with bGH doubled its concentration. Although the effects on body and muscle weight were prevented when rats were given anti-rGH and bGH simultaneously, the possibility of mediation by other hormones cannot be precluded.

Several insulin-like growth factor-I analogues and complexes of insulin-like growth factors-I and -II with insulin-like growth factor-binding protein-3 fail to mimic the effect of growth hormone upon lactation in the rat.

Lactation was suppressed in rats using a combined treatment of bromocriptine (to reduce prolactin concentrations) and a specific antiserum to rat GH administered twice daily for 2 days. When milk production had ceased, as determined by litter weight loss and the absence of milk in the stomachs of pups, attempts were made to reinitiate lactation using prolactin, GH, insulin-like growth factor-I (IGF-I) precomplexed to recombinant human IGF-binding protein-3 (hIGFBP-3) or IGF-I plus IGF-II precomplexed to hIGFBP-3. Despite the fact that all treatments except prolactin led to increases in serum IGFs and IGFBP-3, only prolactin and GH provoked the reinitiation of milk production as determined by increased litter weight gain, milk in the stomach of pups and a significant increase in the weight of the mammary glands. Since the mammary gland has been shown to produce IGFBPs which may inhibit IGF action we also tested three IGF-I analogues, R3-IGF-I, Long-IGF-I and Long-R3-IGF-I. R3-IGF-I has a single amino acid substitution (Glu to Arg) at position 3 whereas Long-IGF-I has a 13 amino acid N-terminal extension. These modifications dramatically reduce the ability of these analogues to bind to IGFBPs although they remain active at the IGF-I receptor. Such IGF analogues would therefore be expected to be active irrespective of the production of inhibitory IGFBPs. However, none was effective in reinitiating lactation, even at doses which have been shown to be biologically effective in terms of nitrogen retention.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased fat content and increased lean in pigs treated with antibodies to adipocyte plasma membranes.

Antibodies were prepared in sheep against purified plasma membranes from pig adipocytes. Western (immuno) blotting revealed reactions of the antisera with a large number of proteins in adipocyte plasma membranes but remarkably few in plasma membranes from muscle, kidney, liver, lung, brain, spleen, and erythrocytes. This illustrated the high degree of specificity the serum had for adipose tissue. When injected into localized subcutaneous sites such antisera were able to cause considerable adipocyte destruction, which resulted in complete loss of adipose tissue from the site for > or = 14 wk. This cell destruction was probably mediated in part by lymphocytic infiltration. Subcutaneous injections were of limited use because of the localized nature of the effects, but, when treatment was administered intraperitoneally, systemic effects were produced that resulted in a 30% reduction in backfat thickness in the region of the last rib and a 25% reduction in fat content of fore- and hind-loin joints that resulted in a significant increase in the percentage of lean tissue. Total feed intake, live weight gain, hot carcass weights, and dressing percentage were unaffected. These results demonstrate the potential for producing long-term reductions in body fat in pigs by an immunization technique that may also provide the unexpected, potential benefit of increased lean deposition. This suggests that fat deposition per se exerts a restrictive influence on lean carcass development.