Vasoinhibin is Generated and Promotes Inflammation in Mild Antigen-induced Arthritis.

Inflammatory arthritis defines a family of diseases influenced by reproductive hormones. Vasoinhibin, a fragment of the hormone prolactin (PRL), has antiangiogenic and proinflammatory properties. We recently showed that vasoinhibin reduces joint inflammation and bone loss in severe antigen-induced arthritis (AIA) by an indirect mechanism involving the inhibition of pannus vascularization. This unexpected finding led us to hypothesize that a severe level of inflammation in AIA obscured the direct proinflammatory action of vasoinhibin while allowing the indirect anti-inflammatory effect via its antiangiogenic properties. In agreement with this hypothesis, here we show that the intra-articular injection of an adeno-associated virus type-2 vector encoding vasoinhibin reduced joint inflammation in a severe AIA condition, but elevated joint inflammation in a mild AIA model. The proinflammatory effect, unmasked in mild AIA, resulted in joint swelling, enhanced leukocyte infiltration, and upregulation of expression of genes encoding proinflammatory mediators (Il1b, Il6, Inos, Mmp3), adhesion molecule (Icam1), and chemokines (Cxcl1, Cxcl2, Cxcl3, Ccl2). Furthermore, vasoinhibin induced the expression of proinflammatory mediators and chemokines in cultured synovial fibroblasts through nuclear factor-κB. Finally, matrix metalloproteases and cathepsin D, upregulated in the arthritic joint, cleaved PRL to vasoinhibin, and vasoinhibin levels increased in the circulation of mice subjected to AIA. We suggest that vasoinhibin is generated during inflammatory arthritis and acts on synovial fibroblasts and endothelial cells to initially promote and later inhibit inflammation, respectively. These opposite effects may work together to help keep joint inflammation under balance.

Mitochondrial Oxidation of the Cytoplasmic Reducing Equivalents at the Onset of Oxidant Stress in the Isoproterenol-Induced Rat Myocardial Infarction.

We have developed and characterized a model of isoproterenol (ISO)-induced myocardial necrosis, identifying three stages of cardiac damage: a pre-infarction (0-12 h), infarction (24 h), and post-infarction period (48-96 h). Using this model, we have previously found alterations in calcium homeostasis and their relationship with oxidant stress in mitochondria, which showed deficient oxygen consumption and coupled ATP synthesis. Therefore, the present study was aimed at assessing the mitochondrial ability to transport and oxidize cytoplasmic reducing equivalents (NADH), correlating the kinetic parameters of the malate-aspartate shuttle, oxidant stress, and mitochondrial functionality. Our results showed only discreet effects during the cardiotoxic ISO action on the endogenous malate-aspartate shuttle activity, suggesting that endogenous mitochondrial NADH oxidation capacity (Nohl dehydrogenase) was not affected by the cellular stress. On the contrary, the reconstituted system showed significant enhancement in maximal capacity of the malate-aspartate shuttle activity only at later times (post-infarction period), probably as a compensatory part of cardiomyocytes' response to the metabolic and functional consequences of the infarcted tissue. Therefore, these findings support the notion that heart damage associated with myocardial infarction suffers a set of sequential biochemical and metabolic modifications within cardiomyocytes, where mitochondrial activity, controlling the redox state, could play a relevant role.

Impaired prolactin actions mediate altered offspring metabolism induced by maternal high-fat feeding during lactation.

Maternal diet during lactation affects offspring metabolic health throughout life. Prolactin (PRL) is present in high quantities in maternal milk; however, the effects of milk PRL on the offspring remain poorly characterized. In this study, we evaluated whether feeding a high-fat diet (HFD) to rats during lactation alters PRL, both in the mother's serum and in milk, and whether this factor contributes to HFD-induced metabolic dysfunction in the offspring. Maternal HFD resulted in decreased PRL levels in milk (but not in serum), reduced mammary gland (MG) PRL receptor expression, and altered MG structure and function. Offspring from HFD-fed dams had increased body weight and adiposity, and developed fatty liver, hyperinsulinemia, and insulin resistance at weaning. Increasing PRL levels in the HFD-fed mothers by subcutaneous osmotic minipumps releasing PRL normalized MG function and PRL levels in milk. Moreover, PRL treatment in HFD-fed mothers, or directly in their pups via oral PRL administration, increased liver STAT5 phosphorylation, reduced visceral adiposity, ameliorated fatty liver, and improved insulin sensitivity in offspring. Our results show that HFD impairs PRL actions during lactation to negatively affect MG physiology and directly impair offspring metabolism.-De los Ríos, E. A., Ruiz-Herrera, X., Tinoco-Pantoja, V., López-Barrera, F., Martínez de la Escalera, G., Clapp, C., Macotela, Y. Impaired prolactin actions mediate altered offspring metabolism induced by maternal high-fat feeding during lactation.

Prolactin blocks the expression of receptor activator of nuclear factor κB ligand and reduces osteoclastogenesis and bone loss in murine inflammatory arthritis.

BACKGROUND: Prolactin (PRL) reduces joint inflammation, pannus formation, and bone destruction in rats with polyarticular adjuvant-induced arthritis (AIA). Here, we investigate the mechanism of PRL protection against bone loss in AIA and in monoarticular AIA (MAIA). METHODS: Joint inflammation, trabecular bone loss, and osteoclastogenesis were evaluated in rats with AIA treated with PRL (via osmotic minipumps) and in mice with MAIA that were null (Prlr-/-) or not (Prlr+/+) for the PRL receptor. To help define target cells, synovial fibroblasts from Prlr+/+ mice were treated or not with proinflammatory cytokines ((Cyt), including TNFα, IL-1ß, and interferon (IFN)γ) with or without PRL, and these synovial cells were co-cultured or not with bone marrow osteoclast progenitors from Prlr+/+ or Prlr-/- mice. RESULTS: In AIA, PRL treatment reduced joint swelling, increased trabecular bone area, lowered osteoclast density, and reduced mRNA levels of osteoclast-associated genes (tartrate-resistant acid phosphatase (Trap)), cathepsin K (Ctsk), matrix metalloproteinase 9 (Mmp9), and receptor activator of nuclear factor κB or RANK (Tnfrsf11a)), of genes encoding cytokines with osteoclastogenic activity (Tnfa, Il1b, Il6, and receptor activator of nuclear factor κB ligand or RANKL (Tnfrsf11)), and of genes encoding for transcription factors and cytokines related to T helper (Th)17 cells (Rora, Rorc, Il17a, Il21, Il22) and to regulatory T cells (Foxp3, Ebi3, Il12a, Tgfb1, Il10). Prlr-/- mice with MAIA showed enhanced joint swelling, reduced trabecular bone area, increased osteoclast density, and elevated expression of Tnfa, Il1b, Il6, Trap, Tnfrsf11a, Tnfrsf11, Il17a, Il21, Il22, 1 l23, Foxp3, and Il10. The expression of the long PRL receptor form increased in arthritic joints, and in synovial membranes and cultured synovial fibroblasts treated with Cyt. PRL induced the phosphorylation/activation of signal transducer and activator of transcription-3 (STAT3) and inhibited the Cyt-induced expression of Il1b, Il6, and Tnfrsf11 in synovial fibroblast cultures. The STAT3 inhibitor S31-201 blocked inhibition of Tnfrsf11 by PRL. Finally, PRL acted on both synovial fibroblasts and osteoclast precursor cells to downregulate Cyt-induced osteoclast differentiation. CONCLUSION: PRL protects against osteoclastogenesis and bone loss in inflammatory arthritis by inhibiting cytokine-induced expression of RANKL in joints and synovial fibroblasts via its canonical STAT3 signaling pathway.

Prolactin Promotes Adipose Tissue Fitness and Insulin Sensitivity in Obese Males.

Excessive accumulation of body fat triggers insulin resistance and features of the metabolic syndrome. Recently, evidence has accumulated that obesity, type 2 diabetes, and metabolic syndrome are associated with reduced levels of serum prolactin (PRL) in humans and rodents, raising the question of whether low PRL levels contribute to metabolic dysfunction. Here, we have addressed this question by investigating the role of PRL in insulin sensitivity and adipose tissue fitness in obese rodents and humans. In diet-induced obese rats, treatment with PRL delivered via osmotic mini-pumps, improved insulin sensitivity, prevented adipocyte hypertrophy, and reduced inflammatory cytokine expression in visceral fat. PRL also induced increased expression of Pparg and Xbp1s in visceral adipose tissue and elevated circulating adiponectin levels. Conversely, PRL receptor null mice challenged with a high-fat diet developed greater insulin resistance, glucose intolerance, and increased adipocyte hypertrophy compared with wild-type mice. In humans, serum PRL values correlated positively with systemic adiponectin levels and were reduced in insulin-resistant patients. Furthermore, PRL circulating levels and PRL produced by adipose tissue correlated directly with the expression of PPARG, ADIPOQ, and GLUT4 in human visceral and sc adipose tissue. Thus, PRL, acting through its cognate receptors, promotes healthy adipose tissue function and systemic insulin sensitivity. Increasing the levels of PRL in the circulation may have therapeutic potential against obesity-induced metabolic diseases.

Prolactin anterior pituitary expression and circulating levels are reduced in obese and diabetic rats: role of TGF-ß and TNF-α.

The levels of the hormone prolactin (PRL) are reduced in the circulation of patients with Type 2 diabetes and in obese children, and lower systemic PRL levels correlate with an increased prevalence of diabetes and a higher risk of metabolic syndrome. The secretion of anterior pituitary (AP) PRL in metabolic diseases may be influenced by the interplay between transforming growth factor ß (TGF-ß) and tumor necrosis factor α (TNF-α), which inhibit and can stimulate AP PRL synthesis, respectively, and are known contributors to insulin resistance and metabolic complications. Here, we show that TGF-ß and TNF-α antagonize the effect of each other on the expression and release of PRL by the GH4C1 lactotrope cell line. The levels of AP mRNA and circulating PRL decrease in high-fat diet-induced obese rats in parallel with increased and reduced AP levels of TGF-ß and TNF-α mRNA, respectively. Likewise, AP expression and circulating levels of PRL are reduced in streptozotocin-induced diabetic rats and are associated with higher AP expression and protein levels of TGF-ß and TNF-α. The opposing effects of the two cytokines on cultured AP cells, together with their altered expression in the AP of obese and diabetic rats suggest they are linked to the reduced PRL production and secretion characteristics of metabolic diseases.

Prolactin promotes cartilage survival and attenuates inflammation in inflammatory arthritis.

Chondrocytes are the only cells in cartilage, and their death by apoptosis contributes to cartilage loss in inflammatory joint diseases, such as rheumatoid arthritis (RA). A putative therapeutic intervention for RA is the inhibition of apoptosis-mediated cartilage degradation. The hormone prolactin (PRL) frequently increases in the circulation of patients with RA, but the role of hyperprolactinemia in disease activity is unclear. Here, we demonstrate that PRL inhibits the apoptosis of cultured chondrocytes in response to a mixture of proinflammatory cytokines (TNF-α, IL-1ß, and IFN-γ) by preventing the induction of p53 and decreasing the BAX/BCL-2 ratio through a NO-independent, JAK2/STAT3-dependent pathway. Local treatment with PRL or increasing PRL circulating levels also prevented chondrocyte apoptosis evoked by injecting cytokines into the knee joints of rats, whereas the proapoptotic effect of cytokines was enhanced in PRL receptor-null (Prlr(-/-)) mice. Moreover, eliciting hyperprolactinemia in rats before or after inducing the adjuvant model of inflammatory arthritis reduced chondrocyte apoptosis, proinflammatory cytokine expression, pannus formation, bone erosion, joint swelling, and pain. These results reveal the protective effect of PRL against inflammation-induced chondrocyte apoptosis and the therapeutic potential of hyperprolactinemia to reduce permanent joint damage and inflammation in RA.

Prolactin promotes normal liver growth, survival, and regeneration in rodents: effects on hepatic IL-6, suppressor of cytokine signaling-3, and angiogenesis.

Prolactin (PRL) is a potent liver mitogen and proangiogenic hormone. Here, we used hyperprolactinemic rats and PRL receptor-null mice (PRLR(-/-)) to study the effect of PRL on liver growth and angiogenesis before and after partial hepatectomy (PH). Liver-to-body weight ratio (LBW), hepatocyte and sinusoidal endothelial cell (SEC) proliferation, and hepatic expression of VEGF were measured before and after PH in hyperprolactinemic rats, generated by placing two anterior pituitary glands (AP) under the kidney capsule. Also, LBW and hepatic expression of IL-6, as well as suppressor of cytokine signaling-3 (SOCS-3), were evaluated in wild-type and PRLR(-/-) mice before and after PH. Hyperprolactinemia increased the LBW, the proliferation of hepatocytes and SECs, and VEGF hepatic expression. Also, liver regeneration was increased in AP-grafted rats and was accompanied by elevated hepatocyte and SEC proliferation, and VEGF expression compared with nongrafted controls. Lowering circulating PRL levels with CB-154, an inhibitor of AP PRL secretion, prevented AP-induced stimulation of liver growth. Relative to wild-type animals, PRLR(-/-) mice had smaller livers, and soon after PH, they displayed an approximately twofold increased mortality and elevated and reduced hepatic IL-6 and SOCS-3 expression, respectively. However, liver regeneration was improved in surviving PRLR(-/-) mice. PRL stimulates normal liver growth, promotes survival, and regulates liver regeneration by mechanisms that may include hepatic downregulation of IL-6 and upregulation of SOCS-3, increased hepatocyte proliferation, and angiogenesis. PRL contributes to physiological liver growth and has potential clinical utility for ensuring survival and regulating liver mass in diseases, injuries, or surgery of the liver.

Expression and cellular localization of prolactin and the prolactin receptor in mammalian retina.

Prolactin (PRL), originally associated with milk secretion, is known to have a wide variety of biological actions and diverse sites of production beyond the pituitary gland. Recent studies have demonstrated that PRL is synthesized in retinal tissue. To gain insights into the functional role of PRL in the mammalian retina, we mapped the distribution of the PRL protein and the expression and localization of the PRL receptor (PRLR) in the retina of adult rats and green monkeys. PRL was examined in retinal sections by double immunolabeling combining anti-PRL antibodies with antibodies specific for glutamine synthetase (labeling Müller cells), glial fibrillary acidic protein (labeling astrocytes), or neuronal nuclei protein (labeling neurons). PRL was detected throughout the rat retina: in the photoreceptor outer segments, Müller cells, interneurons, ganglion cells, and astrocytes. The PRLR was examined by RT-PCR, in situ hybridization, immunohistochemistry, and Western blot. The long isoform of the PRLR was localized in the photoreceptor nuclear layer, inner nuclear layer, and ganglion cell layer of rat retina. The monkey retina showed a similar distribution of PRL and PRLR immunoreactivities. These findings suggest that PRL functions as a local regulator of various cell types in the mammalian retina.

Matrix metalloproteases from chondrocytes generate an antiangiogenic 16 kDa prolactin.

The 16 kDa N-terminal fragment of prolactin (16K-prolactin) is a potent antiangiogenic factor. Here, we demonstrate that matrix metalloproteases (MMPs) produced and secreted by chondrocytes generate biologically functional 16K-prolactin from full-length prolactin. When incubated with human prolactin at neutral pH, chondrocyte extracts and conditioned medium, as well as chondrocytes in culture, cleaved the Ser155-Leu156 peptide bond in prolactin, yielding - upon reduction of intramolecular disulfide bonds - a 16 kDa N-terminal fragment. This 16K-prolactin inhibited basic fibroblast growth factor (FGF)-induced endothelial cell proliferation in vitro. The Ser155-Leu156 site is highly conserved, and both human and rat prolactin were cleaved at this site by chondrocytes from either species. Conversion of prolactin to 16K-prolactin by chondrocyte lysates was completely abolished by the MMP inhibitors EDTA, GM6001 or 1,10-phenanthroline. Purified MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 cleaved human prolactin at Gln157, one residue downstream from the chondrocyte protease cleavage site, with the following relative potency: MMP-8 > MMP-13 > MMP-3 > MMP-1= MMP-2 > MMP-9. Finally, chondrocytes expressed prolactin mRNA (as revealed by RT-PCR) and they contained and released antiangiogenic N-terminal 16 kDa prolactin (detected by western blot and endothelial cell proliferation). These results suggest that several matrix metalloproteases in cartilage generate antiangiogenic 16K-prolactin from systemically derived or locally produced prolactin.

Opposite association of serum prolactin and survival in patients with colon and rectal carcinomas: influence of preoperative radiotherapy.

Prolactin (PRL) is a pleiotropic hormone associated with the progression of various cancers, including colorectal cancer (CRC). Here we investigate whether the association of serum PRL concentration and survival is affected by tumor location and preoperative radiotherapy (PRERT) in patients with CRC cancer. Serum PRL was determined in 82 CRC patients without previous treatment. Patients with PRL concentrations at and above the 75th percentile (high PRL) or below this level (low PRL), had a significant correlation with overall survival determined using the Kaplan-Meier method. In colon cancer, there was an increased risk of mortality when PRL values were at and above the highest quartile (22% vs. 73%; P = 0.01). In contrast, in rectal cancer, high PRL values were associated with a significant overall survival advantage (88% vs. 44%; P = 0.05), which became more significant (100% vs. 34%; P = 0.005) when only rectal cancer patients receiving PRERT were compared. These findings suggest that tumor location and adjuvant radiotherapy influence the association between circulating PRL and survival in CRC.

Prolactins are natural inhibitors of angiogenesis in the retina.

PURPOSE: Disruption of the normally antiangiogenic environment of the retina leads to aberrant angiogenesis, the major cause of vision loss throughout the world. Prolactin (PRL), the hormone originally associated with milk production, can be proteolytically processed to 16K-PRL, a 16 kDa N-terminal PRL fragment with potent antiangiogenic and vasoconstrictive actions. This study was conducted to determine whether 16K-PRL is found naturally in the retina and plays a role in angiogenesis and vasodilation. METHODS: Expression of PRL mRNA in rat retina was determined by RT-PCR and in situ hybridization. Western blot was used to examine the expression of PRL and derived fragments in retinal homogenates. The role of PRL and 16K-PRL in the retina was studied by intravitreal injection of either antibodies against PRL or small interfering RNAs (siRNA), to suppress expression of retinal PRL mRNA. RESULTS: Rat retina expressed PRL mRNA in the outer nuclear, outer plexiform, inner nuclear, and ganglion cell layers. Both full-length PRL and N-terminal 16K-PRL were detected in retinal homogenates by polyclonal and monoclonal antibodies. The intravitreal injection of antibodies able to neutralize the actions of 16K-PRL increased the number of retinal blood vessels and capillary area by threefold. Furthermore, siRNA-mediated inhibition of PRL mRNA increased retinal neovascularization threefold and resulted in a significant increase in vasodilation. CONCLUSIONS: These results demonstrate that PRL is synthesized and cleaved to antiangiogenic 16K-PRL by retinal tissue and that these molecules play a key role in preventing angiogenesis in the healthy retina.

Prolactin stimulates integrin-mediated adhesion of circulating mononuclear cells to endothelial cells.

Attachment of leukocytes to endothelial cells is an essential step for the extravasation and recruitment of cells at sites of inflammation. The pituitary hormone prolactin (PRL) is involved in the inflammatory process. Here, we show that treatment with PRL of human peripheral blood mononuclear cells (PBMC) stimulates their adhesion to human umbilical vein endothelial cells (HUVEC) activated by interleukin-1beta. Stimulation of adhesion by PRL is mediated via integrins leukocyte functional antigen-1 (LFA-1) and very late antigen-4 (VLA-4), because immunoneutralization of both integrins prevents PRL action. Also, PRL promotes the adhesion of PBMC to immobilized intercellular adhesion molecule-1 and fibronectin, ligands for LFA-1 and VLA-4, respectively. Stimulation of integrin-mediated cell adhesion by PRL may involve the activation of chemokine receptors, because PRL upregulates the expression of the G-protein-coupled chemokine receptor CXCR3 in PBMC, and pertussis toxin, a specific G-protein inhibitor, blocks PRL stimulation of PBMC adhesion to HUVEC. In addition, PRL stimulates tyrosine phosphorylation pathways leading to leukocyte adhesion. PRL triggered the tyrosine phosphorylation of Janus kinase-2, of signal transducer and activator of transcription-3 and 5, and of the focal adhesion protein paxillin. Furthermore, genistein, a tyrosine kinase inhibitor, blocked PRL-stimulated adhesion of PBMC and Jurkat T-cells to HUVEC. These results suggest that PRL promotes integrin-mediated leukocyte adhesion to endothelial cells via chemokine receptors and tyrosine phosphorylation signaling pathways.

16K-prolactin inhibits activation of endothelial nitric oxide synthase, intracellular calcium mobilization, and endothelium-dependent vasorelaxation.

Activation of endothelial nitric oxide synthase (eNOS) and subsequent nitric oxide production (NO) are events that mediate the effect of important angiogenic, vasopermeability, and vasorelaxation factors, including vascular endothelial growth factor (VEGF), bradykinin (BK), and acetylcholine (ACh). The N-terminal 16-kDa fragment of prolactin (16K-PRL) acts on endothelial cells to inhibit angiogenesis both in vivo and in vitro. Here, we show that 16K-PRL inhibits VEGF-induced eNOS activation in endothelial cells. Inhibition of eNOS activation may mediate the antiangiogenic properties of 16K-PRL, because the NO donor (Z)-1-[2-(2-aminoethyl)- N-(2-ammonio-ethyl)amino]diazen-1-ium-1,2-diolate (DETANONOate) prevented 16K-PRL from blocking the VEGF-induced proliferation of endothelial cells. In addition, 16K-PRL inhibited eNOS activation by BK and blocked the BK-evoked transient increase in intracellular Ca(2+) in endothelial cells. This finding suggests that 16K-PRL interferes with the mobilization of intracellular Ca(2+), thereby inhibiting the Ca(2+)-dependent activation of eNOS. Blockage of eNOS activation can lead to inhibition of vasodilation. Consistently, 16K-PRL inhibited BK-induced relaxation of coronary vessels in isolated perfused guinea pig hearts. Moreover, 16K-PRL inhibited eNOS activation induced by ACh, and this action resulted in the inhibition of both ACh-evoked relaxation of coronary vessels in isolated perfused rat hearts and ACh-induced, endothelium-dependent relaxation of rat aortic segments. In conclusion, 16K-PRL can block the Ca(2+)-mediated activation of eNOS by three different vasoactive substances, and this action results in the inhibition of both angiogenesis and vasorelaxation.

Prolactin in eyes of patients with retinopathy of prematurity: implications for vascular regression.

PURPOSE: Disruption of the anti-angiogenic environment of the retina leads to neovascular eye diseases, including retinopathy of prematurity (ROP). Prolactin (PRL), the hormone originally associated with milk secretion, is proteolytically processed to 16K-PRL, a fragment with potent antiangiogenic, proapoptotic effects. Whether 16K-PRL is produced in eyes of patients with ROP and promotes the regression of intraocular blood vessels associated with this disease was investigated. METHODS: PRL was quantified in the aqueous humor, subretinal fluid, and serum from patients with stage 5 ROP and in patients with non-neovascular eye disorders. Intraocular expression of PRL was evaluated by RT-PCR, in situ hybridization, and Western blot analysis. AntiPRL antibodies were injected intravitreously in neonatal rats, and apoptosis of hyaloid vessels determined by TUNEL and ELISA. RESULTS: PRL was elevated in ocular fluids and serum from ROP patients. There was no correlation between PRL in ocular fluids and its level in serum, whereas PRL in aqueous humor and subretinal fluid were significantly correlated. PRL mRNA was expressed in blood vessels and leukocytes within retrolental fibrovascular membranes of ROP patients, and these membranes contained a 16 kDa immunoreactive PRL. The 16K-PRL isoform was more concentrated in subretinal fluid than in serum and was generated from PRL by subretinal fluid proteases. Intravitreous injection of neutralizing antiPRL antibodies inhibited the apoptosis of hyaloid vessels in neonatal rats. CONCLUSIONS: 16K-PRL derived from PRL internalized from the circulation or synthesized intraocularly can stimulate apoptosis-induced vascular regression and contribute to the development and progression of ROP.

Hypoxia inhibits expression of prolactin and secretion of cathepsin-D by the GH4C1 pituitary adenoma cell line.

Diminished oxygen concentration within growing tumors may stimulate neovascularization by inducing both up-regulation of angiogenic factors and down-regulation of antiangiogenic agents. A potentially important molecule in the growth of pituitary adenomas is prolactin (PRL), which can be cleaved by cathepsin-D to yield a 16-kDa form (16K-PRL) with potent antiangiogenic effects. We examined the expression of PRL in cultured GH4C1 pituitary adenoma cells after exposure to hypoxia (0.1% oxygen) for periods of 12 to 36 hours. In contrast to increased expression of the angiogenic factor vascular endothelial growth factor in hypoxic cells, PRL mRNA and levels of intracellular and secreted PRL were significantly reduced under hypoxia. The reduction was not attributable to a general suppression of either transcription or protein synthesis. Although 16K-PRL was not evident in conditioned medium at physiologic pH, lowering the pH to mimic the acidic tumor microenvironment resulted in generation of 16K-PRL, which was sharply reduced in medium drawn from hypoxic cells. Production of 16K-PRL was blocked by the cathepsin-D inhibitor pepstatin-A, and the reduced 16K-PRL formation in hypoxic-conditioned medium correlated with a decrease in secretion of cathepsin-D and its precursor, procathepsin-D. Thus, hypoxia acts upon GH4C1 cells to increase vascular endothelial growth factor expression, decrease PRL synthesis, and suppress conversion of PRL to 16K-PRL via inhibition of cathepsin-D proteolysis. These mechanisms may act in concert to stimulate angiogenesis in prolactinomas.