scholarly journals Expression of glucose-dependent insulinotropic polypeptide in the zebrafish

2009 ◽  
Vol 297 (6) ◽  
pp. R1803-R1812 ◽  
Author(s):  
Michelle C. Musson ◽  
Lisa I. Jepeal ◽  
Patrick D. Mabray ◽  
Irina V. Zhdanova ◽  
Wellington V. Cardoso ◽  
...  
Keyword(s):  
Early Stage ◽  
Model Organism ◽  
Receptor Activation ◽  
Evolutionary Development ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Glucagon Receptor ◽  
Nutrient Deposition ◽  
Zebrafish Larvae

In mammals, glucose-dependent insulinotropic polypeptide (GIP) is synthesized predominately in the small intestine and functions in conjunction with insulin to promote nutrient deposition. However, little is known regarding GIP expression and function in early vertebrates like the zebrafish, a model organism representing an early stage in the evolutionary development of the compound vertebrate pancreas. Analysis of GIP and insulin ( insa) expression in zebrafish larvae by RT-PCR demonstrated that although insa was detected as early as 24 h postfertilization (hpf), GIP expression was not demonstrated until 72 hpf, shortly after the completion of endocrine pancreatic development but prior to the commencement of independent feeding. Furthermore, whole mount in situ hybridization of zebrafish larvae showed expression of GIP and insa in the same tissues, and in adult zebrafish, RT-PCR and immunohistochemistry demonstrated GIP expression in both the intestine and the pancreas. Receptor activation studies showed that zebrafish GIP was capable of activating the rat GIP receptor. Although previous studies have identified four receptors with glucagon receptor-like sequences in the zebrafish, one of which possesses the capacity to bind GIP, a functional analysis of these receptors has not been performed. This study demonstrates interactions between the latter receptor and zebrafish GIP, identifying it as a potential in vivo target for the ligand. Finally, food deprivation studies in larvae demonstrated an increase in GIP and proglucagon II mRNA levels in response to fasting. In conclusion, the results of these studies suggest that although the zebrafish appears to be a model of an early stage of evolutionary development of GIP expression, the peptide may not possess incretin properties in this species.

scholarly journals Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas

2002 ◽  
pp. 795-802 ◽  
Author(s):  
F Fallo ◽  
V Pezzi ◽  
L Barzon ◽  
P Mulatero ◽  
F Veglio ◽  
...  
Keyword(s):  
Real Time ◽  
Secretory Activity ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Pathophysiological Role ◽  
Aldosterone Production

BACKGROUND: The presence and pathophysiological role of CYP11B1 (11beta-hydroxylase) gene in the zona glomerulosa of human adrenal cortex is still controversial. METHODS: In order to specifically quantify CYP11B1, CYP11B2 (aldosterone synthase) and CYP17(17alpha-hydroxylase) mRNA levels, we developed a real-time RT-PCR assay and examined the expression in a series of adrenal tIssues, including six normal adrenals from patients adrenalectomized for renal cancer and twelve aldosterone-producing adenomas (APA) from patients with primary aldosteronism. RESULTS: CYP11B1 mRNA levels were clearly detected in normal adrenals, which comprised both zona glomerulosa and fasciculata/reticularis cells, but were also measured at a lower range (P<0.05) in APA. The levels of CYP11B2 mRNA were lower (P<0.005) in normal adrenals than in APA. CYP17 mRNAlevels were similar in normal adrenals and in APA. In patients with APA, CYP11B2 and CYP11B1 mRNA levels were not correlated either with basal aldosterone or with the change from basal aldosterone in response to posture or to dexamethasone. No correlation between CYP11B1 mRNA or CYP11B2 mRNA and the percentage of zona fasciculata-like cells was observed in APA. CONCLUSIONS: Real-time RT-PCR can be reliably used to quantify CYP11B1 and CYP11B2 mRNA levels in adrenal tIssues. Expression of CYP11B1 in hyperfunctioning zona glomerulosa suggests an additional formation of corticosterone via 11beta-hydroxylase, providing further substrate for aldosterone biosynthesis. CYP11B1 and CYP11B2 mRNA levels in APA are not related to the in vivo secretory activity of glomerulosa cells, where post-transcriptional factors might ultimately regulate aldosterone production.

scholarly journals Altered Expression of Three EGFR Posttranslational Regulators MDGI, MIG6, and EIG121 in Invasive Breast Carcinomas

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Didier Meseure ◽  
Kinan Drak Alsibai ◽  
Sophie Vacher ◽  
Rana Hatem ◽  
Andre Nicolas ◽  
...  
Keyword(s):  
Expression Patterns ◽  
Prognostic Significance ◽  
Growth Factor Receptor ◽  
Growth Inhibitor ◽  
Receptor Activation ◽  
Mrna Levels ◽  
Breast Carcinomas ◽  
Rt Pcr ◽  
Altered Expression ◽  
Invasive Breast Carcinomas

Epidermal growth factor receptor (EGFR) signalling is a highly regulated process with a tight balance between receptor activation and inactivation in invasive breast carcinomas (IBCs) particularly in triple-negative carcinomas (TNC). Clinical trials using anti-EGFR therapies are actually performed although no activating alterations (mutations, amplifications, or rearrangements) of EGFR have been clearly recognized in order to identify new targeted modalities for IBCs. We explored mammary-derived growth inhibitor (MDGI), estrogen-induced gene-121 (EIG121), and mitogen-induced gene-6 (MIG6), three posttranslational EGFR trafficking molecules implicated in EGFR spatiotemporal regulatory pathway. We quantified MDGI, EIG121, and MIG6 at mRNA levels by using real-time quantitative RT-PCR in a series of 440 IBCs and at protein levels by using immunohistochemistry in a series of 88 IBCs. Results obtained by RT-PCR showed that in IBCs, MDGI, MIG6, and EIG121 mRNA were mainly underexpressed (25.7%, 45.0%, and 16.1%, respectively) particularly in the TNC subtype for EIG121 (60.3%). We also observed mRNA overexpression of MDGI and EIG121, respectively, in 12.7% and 22.3% of IBCs. These altered mRNA expressions were confirmed at the protein level. Some links were found between expression patterns of these three genes and several classical pathological and clinical parameters. Only EIG121 was found to have a prognostic significance (p=0.0038). Altered expression of these three major EGFR posttranslational negative regulators could create an aberrant EGFR-mediated oncogenic signalling pathway in IBCs. MDGI, MIG6, and EIG121 expression status also may be potential useful biomarkers (sensitivity or resistance) in targeted EGFR therapy.

Feasibility and Efficacy of Mobilization of BCR/ABL- PBSC in CML Patients Receiving Imatinib.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2858-2858
Author(s):  
Ravi Bhatia ◽  
Helen Xu ◽  
David Snyder ◽  
Tinisha McDonald ◽  
Marilyn Slovak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Median Time ◽  
Autologous Transplantation ◽  
Disease Stage ◽  
Imatinib Treatment ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Target Number ◽  
Cd34 Cells

Abstract Although imatinib is highly effective in inducing remissions in CML patients, the long-term durability of response is not clear. Here we report updated results of a clinical trial investigating the feasibility and efficacy of collection and storage of PBSC from patients in complete cytogenetic remission (CCR) on imatinib, for use in autologous transplantation in the event of subsequent relapse. PBSC were collected from 36 patients [31 CP, 5 AP (at start of imatinib); median age 45 years (range 22–70); 21 males, 15 females; median time from diagnosis 25 months (mos) (6–90); median duration of imatinib treatment 13 mos (6–41); median time from CCR 7 mos (1–26)]. Patients were administered G-CSF (10μg/kg/day), and PBSC collection initiated on day +5 with a targeted minimum of 2x106 CD34+ cells/kg. Imatinib was continued during G-CSF administration and PBSC collection. The G-CSF dose was escalated in case of poor collection. The median number of CD34+ cells (106/kg) collected was 2.56 (0.31–6.19) with a median of 3 phereses (1–13). Five patients failed to collect the target number of CD34+ cells, achieving a median of 0.87x106 CD34+ cells after a median 5 collections. Seven patients required &gt;6 collections to reach the target cell dose. There was no significant relationship between rapid (≤3) or slow (&gt;3) collection, or failure to collect, and clinical characteristics such as age, sex, disease stage, prior interferon, time from diagnosis, time on imatinib, and duration of CCR. PBSC collections were evaluated for BCR/ABL contamination by cytogenetics and PCR (Q-PCR and nested RT-PCR). Ph+ cells were detected on cytogenetic examination in 1 or more collections from 5 patients and Ph- abnormal clones were detected in 4 patients. Patients with Ph+ PBSC were slower collectors than those with Ph- PBSC (median 8 vs. 3 collections, p=0.04). BCR/ABL mRNA was detected by PCR in 1 or more collection from 30 of 32 patients evaluated. Two patients, both with BCR/ABL mRNA detected in pre-mobilzation marrow, collected with a single PCR negative collection. Three patients had collections with low levels of BCR/ABL mRNA detected only with more sensitive nested RT-PCR. Of 134 separate collections analyzed, BCR/ABL mRNA was detected by Q-PCR in 113 (84%), by nested RT-PCR in 11 (8%), while 10 (8%) were PCR negative. Rapid collectors had significantly lower BCR/ABL mRNA levels in their collections compared to pre-mobilization marrow (p&lt;0.05), whereas slow collectors did not show significant change in BCR/ABL levels. CD34+ cells isolated from PBSC showed significantly increased BCR/ABL mRNA levels compared with total nucleated cells (BCR/ABL:B2M ratio of 0.0006±0.0002 for NC vs. 0.03±0.01 for CD34+ cells, p=0.002). PBSC were injected into NOD/SCID mice to evaluate for presence of BCR/ABL+ progenitors capable of in vivo engraftment. Human cell engraftment was confirmed by flow cytometry in 3 of 4 patients, and BCR/ABL mRNA was detected in engrafted cells by Q-PCR. Our results indicate that cytogenetically negative PBSC collections can be obtained from CML patients receiving imatinib, but that mobilization is relatively poor. Rapid collectors have reduced BCR/ABL+ cell contamination in PBSC collections, and PCR negative collections are possible. However, the majority of PBSC products show evidence of persistent malignant stem cells. Additional strategies to enhance reliability and rapidity of collection and further deplete BCR/ABL+ stem cells in the PBSC product need to be explored.

Protease-Activated Receptor 1 (PAR-1) Is Required and Rate-Limiting for Thrombin-Enhanced Experimental Pulmonary Metastasis

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3694-3700 ◽  
Author(s):  
Mary Lynn Nierodzik ◽  
Kui Chen ◽  
Kenichi Takeshita ◽  
Jian-Jun Li ◽  
Yao-Qi Huang ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Pulmonary Metastasis ◽  
Receptor Activation ◽  
Northern Analysis ◽  
Wild Type ◽  
Rt Pcr ◽  
In Vivo Experiments ◽  
B16f10 Cells ◽  
Empty Plasmid

Thrombin-treated tumor cells induce a metastatic phenotype in experimental pulmonary murine metastasis. Thrombin binds to a unique protease-activated receptor (PAR-1) that requires N-terminal proteolytic cleavage for activation by its tethered end. A 14-mer thrombin receptor activation peptide (TRAP) of the tethered end induces the same cellular changes as thrombin. Four murine tumor cells (Lewis lung, CT26 colon CA, B16F10 melanoma, and CCL163 fibroblasts) contain PAR-1, as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). B16F10 cells did not contain the two other thrombin receptors, PAR-3 and glycoprotein Ib. TRAP-treated B16F10 tumor cells enhance pulmonary metastasis 41- to 48-fold (n = 17). Thrombin-treated B16F10 cells transfected with full-length murine PAR-1 sense cDNA (S6, S7, S14, and S22) enhanced their adhesion to fibronectin 1.5- to 2.4-fold (n = 5, P < .04), whereas thrombin-treated wild-type cells do not. S6 (adhesion index, 1.5-fold) and S14 (index, 2.4-fold) when examined by RT-PCR and Northern analysis showed minimal expression of PAR-1 for S6 over wild-type and considerable expression for S14. Immunohistochemistry showed greater expression of PAR-1 for S14 compared with wild-type or empty-plasmid transfected cells. In vivo experiments with the thrombin-treated S14 transfectant showed a fivefold to sixfold increase in metastases compared with empty-plasmid transfected thrombin-treated naive cells or S6 cells (n = 20, P = .0001 to .02). Antisense had no effect on thrombin-stimulated tumor mass. Thus, PAR-1 ligation and expression enhances and regulates tumor metastasis.

scholarly journals Apolipoprotein CI overexpression is not a relevant strategy to block cholesteryl ester transfer protein (CETP) activity in CETP transgenic mice

2004 ◽  
Vol 385 (1) ◽  
pp. 189-195 ◽  
Author(s):  
Thomas GAUTIER ◽  
David MASSON ◽  
Miek C. JONG ◽  
Jean-Paul PAIS DE BARROS ◽  
Linda DUVERNEUIL ◽  
...  
Keyword(s):  
Cholesteryl Ester ◽  
Cholesteryl Ester Transfer Protein ◽  
Mrna Level ◽  
Fold Increase ◽  
Receptor Activation ◽  
Liver X Receptor ◽  
Mrna Levels ◽  
Transfer Protein ◽  
Transfer Activity

ApoCI (apolipoprotein CI) is a potent inhibitor of plasma CETP [CE (cholesteryl ester) transfer protein]. The relevance of apoCI overexpression as a method for CETP blockade in vivo was addressed in the present study in CETPTg/apoCITg mice (mice expressing both human CETP and apoCI). Despite a significant reduction in specific CETP activity in CETPTg/apoCITg mice compared with CETPTg mice [transgenic mouse to human CETP; 46.8±11.1 versus 101.8±25.7 pmol·h−1·(μg of plasma CETP)−1 respectively; P<0.05], apoCI overexpression increased both the CETP mass concentration (3-fold increase; P<0.05) and the hepatic CETP mRNA level (4-fold increase, P<0.005), leading to an increase in total plasma CE transfer activity (by 39%, P<0.05). The ratio of apoB-containing lipoprotein to HDL (high-density lipoprotein) CE was 10-fold higher in CETPTg/apoCITg mice than in apoCITg mice (P<0.0005). It is proposed that the increased CETP expression in CETPTg/apoCITg mice is a direct consequence of liver X receptor activation in response to the accumulation of cholesterol-rich apoB-containing lipoproteins. In support of the latter view, hepatic mRNA levels of other liver X receptor-responsive genes [ABCG5 (ATP-binding cassette transporter GS) and SREBP-1c (sterol-regulatory-binding protein-1c)] were higher in CETPTg/apoCITg mice compared with CETPTg mice. In conclusion, overexpression of apoCI, while producing a significant inhibitory effect on specific CETP activity, does not represent a suitable method for decreasing total CE transfer activity in CETPTg/apoCITg mice, owing to an hyperlipidaemia-mediated effect on CETP gene expression.

scholarly journals OPN Deficiency Increases the Severity of Osteoarthritis Associated with Aberrant Chondrocyte Senescence and Apoptosis and Upregulates the Expression of Osteoarthritis-Associated Genes

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Jian Tian ◽  
Chao Cheng ◽  
Shi-Da Kuang ◽  
Chao Su ◽  
Xin Zhao ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Synovial Fluid ◽  
Joint Damage ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Knee Oa ◽  
Normal Chondrocytes ◽  
Apoptosis Assay ◽  
Oa Chondrocytes

Objectives. A recent work has reported that the elevated osteopontin (OPN) levels in the articular cartilage and synovial fluid are correlated with the progressive osteoarthritis (OA) joint damage, and OPN has a protective effect against OA by suppressing the expressions of OA-associated genes. The present study examined whether the OPN deficiency was susceptible to OA through the regulation of chondrocyte senescence and apoptosis and the expressions of OA-associated genes. Methods. The mRNA levels of COL2A1 and OPN were compared between human OA chondrocytes and normal chondrocytes. The effects of OPN siRNA on the SA-β-Gal expressions and the percentage of apoptotic chondrocytes were examined by using SA-β-Gal staining and apoptosis assay, and the effects on the expressions of COL2A1 and OA-associated genes (COL10A1, IL-1β, TNF-ɑ, MMP-13, and ADAMTS5) were examined by western blot analysis and quantitative real-time RT-PCR. Furthermore, an in vivo OA model was established to examine the effects of OPN siRNA on the senescence and apoptosis of OA chondrocytes and the expressions of OA-associated genes. Results. The mRNA levels of COL2A1 and OPN were decreased in knee OA chondrocytes in comparison with those in normal chondrocytes. The OPN deficiency enhanced the senescence and apoptosis of OA chondrocytes and increased the expressions of COL10A1, IL-1β, TNF-ɑ, MMP-13, and ADAMTS5 but decreased the expression of COL2A1. Meanwhile, OPN deficiency could result in severe, accelerated OA in vivo, which was also associated with enhanced senescence and apoptosis of chondrocytes and elevated expressions of OA-associated genes. Conclusions. The findings of this study suggest that the OPN deficiency can result in accelerated OA, which is associated with enhanced senescence and apoptosis of OA chondrocytes and the upregulated expressions of OA-associated genes.

Gene Expressions Induced by Calcium Phosphate Ceramics after Implantation into the Muscle of Rat

2005 ◽  
Vol 475-479 ◽  
pp. 2363-2366
Author(s):  
Jin Rui Xu ◽  
Hong Song Fan ◽  
Yan Fei Tan ◽  
Xing Dong Zhang
Keyword(s):  
Calcium Phosphate ◽  
Collagen Type ◽  
Early Stage ◽  
Collagen Type I ◽  
Type I ◽  
Rt Pcr ◽  
Gene Expressions ◽  
Porous Hydroxyapatite ◽  
Calcium Phosphate Ceramics

The osteoinductivity of calcium phosphate ceramics has been studied extensively, but the mechanism is still unclear and few reports about the molecular mechanism in the osteoinductive process. In this study the osteoblast related gene expressions induced by biomaterials were investigated by isolating the RNA from the tissue grown in porous hydroxyapatite/tricalcium phosphate (HA/TCP) ceramics implanted in rat femur muscle on day 7, 15, 30, 60, 90,120, and analyzed by RT-PCR technique. RNA extracted from muscle without implant was used as control at the same time. The results showed that osteopontin and osteocalcin genes, the important osteoblastic markers, expressed in early stage, on day 7 after implantation, and were detected at any period. Collagen type I gene expressed on day 60, 90 and 120. It revealed that osteoblast differentiation occurred very early before collagen type I expression after implanting HA/TCP ceramics in vivo.

Role of the TNF pathway in the progression of diabetic nephropathy in KK-Ay mice

2014 ◽  
Vol 306 (11) ◽  
pp. F1335-F1347 ◽  
Author(s):  
Keisuke Omote ◽  
Tomohito Gohda ◽  
Maki Murakoshi ◽  
Yu Sasaki ◽  
Saiko Kazuno ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Early Stage ◽  
Mrna Levels ◽  
Monocyte Chemoattractant Protein 1 ◽  
Protein Levels ◽  
Tnf Α ◽  
Soluble Tnf Receptor

Chronic inflammation promotes the progression of diabetic nephropathy (DN). However, the role of TNF-α remains unclear. The objectives of the present study were to examine whether TNF-α inhibition with a soluble TNF receptor (TNFR)2 fusion protein, i.e., etanercept (ETN), improves the early stage of DN in the type 2 diabetic model of the KK-Ay mouse and to also investigate which TNF pathway, TNFR1 or TNFR2, is predominantly involved in the progression of this disease. ETN was injected intraperitoneally into mice for 8 wk. Renal damage was evaluated by immunohistochemistry, Western blot analysis, and/or real-time PCR. In vitro, mouse tubular proximal cells were stimulated by TNF-α and/or high glucose (HG) and treated with ETN. ETN dramatically improved not only albuminuria but also glycemic control. Renal mRNA and/or protein levels of TNFR2, but not TNF-α and TNFR1, in ETN-treated KK-Ay mice were significantly decreased compared with untreated KK-Ay mice. mRNA levels of ICAM-1, VCAM-1, and monocyte chemoattractant protein-1 and the number of F4/80-positive cells were all decreased after treatment. Numbers of cleaved caspase-3- and TUNEL-positive cells in untreated mice were very few and were not different from ETN-treated mice. In vitro, stimulation with TNF-α or HG markedly increased both mRNA levels of TNFRs, unlike in the in vivo case. Furthermore, ETN partly recovered TNF-α-induced but not HG-induced TNFR mRNA levels. In conclusion, it appears that ETN may improve the progression of the early stage of DN predominantly through inhibition of the anti-inflammatory action of the TNF-α-TNFR2 pathway.

scholarly journals Gene Activation by Cytoplasmic Acidification in Suspension-Cultured Rice Cells in Response to the Potent Elicitor, N-Acetylchitoheptaose

1998 ◽  
Vol 11 (12) ◽  
pp. 1167-1174 ◽  
Author(s):  
Dao-Yao He ◽  
Yoshiaki Yazaki ◽  
Yoko Nishizawa ◽  
Ryota Takai ◽  
Kosumi Yamada ◽  
...  
Keyword(s):  
Propionic Acid ◽  
Plant Cell ◽  
Phenylalanine Ammonia Lyase ◽  
Early Stage ◽  
Gene Activation ◽  
Gene Induction ◽  
Mrna Levels ◽  
Calyculin A ◽  
Cytoplasmic Acidification

N-Acetylchitoheptaose strongly induces a set of defense reactions in suspension-cultured rice cells including cytoplasmic acidification (K. Kuchitsu, Y. Yazaki, K. Sakano, and N. Shibuya, Plant Cell Physiol. 38:1012-1018, 1997) and the accumulation of mRNAs for two rapidly activated genes, EL2 and EL3 (E. Minami, K. Kuchitsu, D.-Y. He, H. Kouchi, N. Midoh, Y. Ohtsuki, and N. Shibuya, Plant Cell Physiol. 37:563-567, 1996), as well as phenylalanine ammonia-lyase (PAL), chitinase and β-1,3-glucanase. Treatment of cells with propionic acid resulted in the accumulation of the mRNAs for EL2, EL3, and PAL in a manner similar to the accumulation induced by N-acetylchitoheptaose. Concomitantly, there was a rapid decrease in the cytoplasmic pH as detected with in vivo 31P-nuclear magnetic resonance (NMR) spectroscopy. Interestingly, K-252a, a potent inhibitor of Ser/Thr protein kinases, strongly inhibited gene induction by N-acetylchitoheptaose, but showed much less inhibition of gene induction caused by propionic acid. Calyculin A, a protein phosphatase inhibitor, induced mRNA accumulations for EL2, EL3 and PAL, with concomitant acidification of the cytoplasm. On the other hand, chitinase and β-glucanase mRNA levels did not change after addition of propionic acid or calyculin A. Treatment of the cells with propionic acid did not induce the production of reactive oxygen species. These results strongly suggest that cytoplasmic acidification at the early stage of elicitor action could be a key step in the signal transduction events leading to the expression of elicitor-responsive genes. A hypothetical model of elicitor signal pathway is proposed based on these results.

scholarly journals Midkine, a Heparin-Binding Growth Factor, Selectively Stimulates Proliferation of Definitive Zone Cells of the Human Fetal Adrenal Gland

2006 ◽  
Vol 91 (10) ◽  
pp. 4050-4056 ◽  
Author(s):  
Hitoshi Ishimoto ◽  
Marcus O. Muench ◽  
Takayuki Higuchi ◽  
Kazuhiro Minegishi ◽  
Mamoru Tanaka ◽  
...  
Keyword(s):  
Growth Factor ◽  
Adrenal Gland ◽  
Real Time ◽  
Outcome Measures ◽  
Mrna Levels ◽  
Heparin Binding ◽  
Rt Pcr ◽  
Pharmacological Interventions

Abstract Context: In the human fetal adrenal gland (HFA), the inner fetal zone (FZ) secretes dehydroepiandrosterone sulfate. The function of the outer definitive zone (DZ) is less clear; however, the DZ phenotype is that of a reservoir of progenitor cells, many of which are mitotically active. Midkine (MK) is a heparin-binding growth factor with various bioactivities. Objective: The objective of this study was to investigate expression, proliferative effects, and ACTH regulation of MK in the HFA. Design and Setting: RNA, cryosections, and primary cell cultures from HFAs (14–24 wk) and adult adrenal RNA were used. Main Outcome Measures: The main outcome measures were MK mRNA levels (measured by quantitative real-time RT-PCR); MK localization (measured by immunostaining); MK proliferative effects and mechanism (measured by proliferation assays, flow cytometry, pharmacological interventions); and ACTH regulation (measured by quantitative real-time RT-PCR). Results: HFA MK mRNA levels were 4-fold higher than in adult adrenals (P &lt; 0.05) and were comparable to levels in fetal and adult brains (positive controls). MK immunoreactivity was abundant throughout the HFA. Exogenous MK caused proliferation of isolated DZ cells but not FZ cells (72 h, P &lt; 0.05). In contrast, basic fibroblast growth factor induced proliferation of cells from both zones. Pharmacological interventions indicated that MK-induced DZ cell proliferation may be mediated by phosphatidylinositol 3-kinase, MAPK kinase, and Src family kinases. ACTH (1 nm) increased MK mRNA by 3.5-fold (48 h, P &lt; 0.01) in isolated FZ cells. Conclusions: MK likely plays a key role in HFA development. MK’s selective in vitro mitotic effects on DZ cells may provide insights into the mechanism underlying the distinct in vivo differences in mitotic activity between the DZ and FZ.

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