Human Fetal Kidney Cells Research Articles

Revealing the spatio-phenotypic patterning of cells in healthy and tumor tissues with mLSR-3D and STAPL-3D.

Despite advances in three-dimensional (3D) imaging, it remains challenging to profile all the cells within a large 3D tissue, including the morphology and organization of the many cell types present. Here, we introduce eight-color, multispectral, large-scale single-cell resolution 3D (mLSR-3D) imaging and image analysis software for the parallelized, deep learning-based segmentation of large numbers of single cells in tissues, called segmentation analysis by parallelization of 3D datasets (STAPL-3D). Applying the method to pediatric Wilms tumor, we extract molecular, spatial and morphological features of millions of cells and reconstruct the tumor’s spatio-phenotypic patterning. In situ population profiling and pseudotime ordering reveals a highly disorganized spatial pattern in Wilms tumor compared to healthy fetal kidney, yet cellular profiles closely resembling human fetal kidney cells could be observed. In addition, we identify previously unreported tumor-specific populations, uniquely characterized by their spatial embedding or morphological attributes. Our results demonstrate the use of combining mLSR-3D and STAPL-3D to generate a comprehensive cellular map of human tumors.

Human fetal kidney cells regenerate acellular porcine kidneys via upregulation of key transcription factors involved in kidney development<em>Running title: Regeneration of porcine kidneys</em>

Interaction between organ-specific stem cells and extracellular matrix (ECM) is crucial for regeneration. We therefore, investigated novel stem cells markers in human kidney and verified the potential of human fetal kidney cells (hFKC) to repopulate decellularized porcine kidneys. Adult and fetal human kidneys were stained by immunohistochemistry for putative stem cell markers. In addition, hFKC were isolated and characterized phenotypically and by gene expression. Furthermore, whole porcine kidneys were decellularized using detergents, cut into 1 mm slices, seeded with hFKC, cultured for 14 days and characterized by histology and qPCR. We found that, decellularized porcine kidneys showed significant loss of DNA but preserved some ECM components. Human fetal kidneys including hFKC expressed stem cell markers CD133, DLK-1, EPCAM and ephrin receptor EphA6. Interestingly, EphA7 and SIX2 were markedly expressed only in fetus. Furthermore, in fetal kidneys EphA7 was co-expressed with DLK-1. Recellularized kidney pieces showed cell infiltration, growing in orchestrated fashion distributed around the scaffold. These pieces also demonstrated cells expressing CK8, CK18, DLK-1, CD133, EphA7, EphB3, PCNA, podocin and increased levels of transcriptional factors in kidney development (SIX2, EYA1, CITED1, LHX1, SALL1, DLK-1 and WT1). We conclude that decellularized porcine kidneys support the culture, proliferation of hFKC and regenerate by upregulation of transcription factors. We suggest that expanded hFKC may be the ideal cell source for whole kidney regeneration in the future. We also postulate EphA7 might be a novel stem cell marker in kidneys.

The 125th Lys and 145th Thr Amino Acids in the GTPase Domain of Goose Mx Confer Its Antiviral Activity against the Tembusu Virus.

The Tembusu virus (TMUV) is an avian pathogenic flavivirus that causes a highly contagious disease and catastrophic losses to the poultry industry. The myxovirus resistance protein (Mx) of innate immune effectors is a key antiviral “workhorse” of the interferon (IFN) system. Although mammalian Mx resistance against myxovirus and retrovirus was witnessed for decades, whether or not bird Mx has anti-flavivirus activity remains unknown. In this study, we found that the transcription of goose Mx (goMx) was obviously driven by TMUV infection, both in vivo and in vitro, and that the titers and copies of TMUV were significantly reduced by goMx overexpression. In both primary (goose embryo fibroblasts, GEFs) and passaged cells (baby hamster kidney cells, BHK21, and human fetal kidney cells, HEK 293T), it was shown that goMx was mainly located in the cytoplasm, and sporadically distributed in the nucleus. The intracellular localization of this protein is attributed to the predicted bipartite nuclear localization signal (NLS; 30 residues: the 441st–471st amino acids of goMx). Intuitively, it seems that the cells with a higher level of goMx expression tend to have lower TMUV loads in the cytoplasm, as determined by an immunofluorescence assay. To further explore the antiviral determinants, a panel of variants was constructed. Two amino acids at the 125th (Lys) and 145th (Thr) positions in GTP-binding elements, not in the L4 loop (40 residues: the 532nd–572nd amino acids of goMx), were vital for the antiviral function of goMx against TMUV in vitro. These findings will contribute to our understanding of the functional significance of the antiviral system in aquatic birds, and the development of goMx could be a valuable therapeutic agent against TMUV.

Calreticulin negatively regulates the surface expression of Cav1.3 L-type calcium channel

BackgroundThe neuroendocrine Cav1.3 L-type Ca channels have been recently found in the Human fetal heart and shown to play a vital role in Ca entry from the sarcolemma into the cell and in Ca homeostasis. Calreticulin, a Ca binding endoplasmic reticulum (ER) resident protein, has been recently shown to translocate to the cell surface where its role and function are just emerging. Here, we demonstrated a novel mechanism of Cav1.3 and calreticulin interaction resulting in downregulation of Cav1.3 channel densities in native Human fetal cardiac cells and Human Embryonic Kidney cell lines (tsA201). Methods and resultsCell surface and cytoplasmic staining of calreticulin was demonstrated first in cultured human fetal cardiomyocytes (HFC), gestational age 18–24weeks, using confocal microscopy thereby establishing that calreticulin is present at the cell surface in HFC. Co-immunoprecipitation from HFC using anti-Cav1.3 Ca channel antibody, and probing with anti-calreticulin antibody revealed a 46kDa band corresponding to calreticulin suggesting that Cav1.3 Ca channel and calreticulin co-assemble in a macromolecular complex. Co-expression of Cav1.3 and calreticulin in tsA201 cells resulted in a decrease in surface expression of Cav1.3 Ca channels. These findings were consistent with the electrophysiological studies showing that co-transfection of Cav1.3 Ca channel and calreticulin resulted in 55% reduction of Cav1.3 Ca current densities recorded from tsA201 cells. ConclusionsThe results show the first evidence that calreticulin: (1) is localized outside the ER on the cell surface of HFC; (2) coimmunoprecipitates with Cav1.3 L-type Ca channel; (3) negatively regulates Cav1.3 surface expression thus resulting in decreased Cav1.3 Ca current densities. The data demonstrate a novel mechanism of modulation of Cav1.3 Ca channel by calreticulin, which may be involved in pathological settings such as autoimmune associated congenital heart block where Cav1.3 Ca channels are downregulated.

Renal Hypodysplasia Associates with a Wnt4 Variant that Causes Aberrant Canonical Wnt Signaling

Abnormal differentiation of the renal stem/progenitor pool into kidney tissue can lead to renal hypodysplasia (RHD), but the underlying causes of RHD are not well understood. In this multicenter study, we identified 20 Israeli pedigrees with isolated familial, nonsyndromic RHD and screened for mutations in candidate genes involved in kidney development, including PAX2, HNF1B, EYA1, SIX1, SIX2, SALL1, GDNF, WNT4, and WT1. In addition to previously reported RHD-causing genes, we found that two affected brothers were heterozygous for a missense variant in the WNT4 gene. Functional analysis of this variant revealed both antagonistic and agonistic canonical WNT stimuli, dependent on cell type. In HEK293 cells, WNT4 inhibited WNT3A induced canonical activation, and the WNT4 variant significantly enhanced this inhibition of the canonical WNT pathway. In contrast, in primary cultures of human fetal kidney cells, which maintain WNT activation and more closely represent WNT signaling in renal progenitors during nephrogenesis, this mutation caused significant loss of function, resulting in diminished canonical WNT/β-catenin signaling. In conclusion, heterozygous WNT4 variants are likely to play a causative role in renal hypodysplasia.

Expression of Stem Cell Markers in the Human Fetal Kidney

In the human fetal kidney (HFK) self-renewing stem cells residing in the metanephric mesenchyme (MM)/blastema are induced to form all cell types of the nephron till 34th week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2) are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24) in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (<10% of HFK cells) and were mostly present within the EpCAMneg and EpCAMdim fractions, indicating putative stem/progenitor markers. In contrast, single markers such as CD24 and CD133 as well as double-positive CD24+CD133+ cells comprise >50% of HFK cells and predominantly co-express EpCAMbright, indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM+EpCAM- and to a lesser extent in NCAM+EpCAM+ fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM+EpCAM+FZD7+), MM stem cells (NCAM+EpCAM-FZD7+) or both (NCAM+FZD7+). These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies.

Cytotoxicity, Antimicrobial Activity and Composition of Essential Oil from Tanacetum balsamita L. Subsp. Balsamita

The aerial parts of Tanacetum balsamita subsp. balsamita were collected at full flowering stage from Tabriz, Iran. The essential oil was isolated by hydrodistillation and analyzed by combination of capillary GC and GC-MS. Twenty seven components were identified, representing 99.2% of the total oil. Carvone (51.0%), beta-thujone (20.8%), 1,8-cineole (4.4%) and alpha-thujone (3.2%) were the main components. The in vitro antimicrobial activity of the essential oil of T. balsamita subsp. balsamita was studied against eight Gram-positive and Gram-negative bacteria (Bacillus subtilis, B. pumulis, Enterococcus faecalis, Staphylococcus aureus, S. epidermidis, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) and three fungi (Candida albicans, Saccharomyces cerevisiae and Aspergillus niger). The results of the antimicrobial activity of the essential oil according to the disc diffusion method and MIC values indicated that the oil exhibited moderate to high antimicrobial activity. The in vitro toxicological study based on the MTT cytotoxicity assay on human fetal skin fibroblast (HFSF) and Monkey kidney (Vero) cell lines showed IC50 values of 2500 and 1250 microg/mL, respectively.

Functional aftereffects of intraparenchymatous injection of human fetal stem and progenitor cells to rats with chronic and acute renal failure

Chronic renal insufficiency was modeled in rats by unilateral nephrectomy and electrocoagulation of both poles of the remaining kidney; acute renal failure was induced by 90-min clamping of the vascular pedicle of the only kidney. Injection of unfractionated culture of human fetal kidney cells or bone marrow mesenchymal stem cells into damaged kidney restored its function in rats with chronic renal insufficiency (observation period up to 2 months). After 2.5 months a relapse of chronic renal insufficiency was observed in 1 of 3 rats receiving human fetal kidney cells and in 1 of 2 animals receiving bone marrow mesenchymal stem cell culture. Injection of bone marrow mesenchymal stem cell culture to rats with acute renal failure improved recovery of renal function and prevented the death from uremia, while injection of total culture of human fetal kidney cells had virtually no effect on the course of acute renal failure.

Alternative Splicing of Human Telomerase Reverse Transcriptase May Not Be Involved in Telomerase Regulation During all-trans-Retinoic Acid-Induced HL-60 Cell Differentiation

Alternative splicing of the human telomerase reverse transcriptase subunit (hTERT) suppresses telomerase activity during the development of human fetal kidney cells into mature cells. Tumor cell differentiation is the process of turning abnormal tumor cells into ‘normal’ cells accompanied by down-regulation of telomerase activity. However, the precise mechanism of the regulation of telomerase activity in differentiated cells is not fully understood. In this study, we observed the role of alternative splicing of hTERT in the regulation of telomerase activity in all-trans-retinoic acid (ATRA)-induced, differentiated HL-60 cells. ATRA-induced down-regulation of telomerase activity in differentiated HL-60 cells was associated with a decrease in hTERT and an increase in human telomerase-associated protein-1 (hTP1) transcription. Expression of full length variant hTERT α+β+ mRNA decreased in a dose- and time-dependent manner. The drop of hTERT β- mRNA was time-dependent. hTERT α- and hTERT α-b- mRNA were reduced dramatically after ATRA treatment. In the dose-effect study, hTERT α+β+ and hTERT β- maintained a relatively stable ratio when telomerase activity decreased largely from treatment with 1 to 5 μM ATRA. Although the splicing pattern of hTERT mRNA was altered in time-effect research, the change was not related to the ATRA-treated decline of telomerase activity. The expression of alternative splicing variants of hTERT also decreased at the protein level. All these results suggested that alternative splicing of hTERT mRNA may not contribute to the suppression of telomerase activity during ATRA-induced HL-60 leukemia cell differentiation.

Inhibition of NF-κB activity by plasmid expressed αMSH peptide

α-Melanocyte Stimulating Hormone (αMSH) is a neuroimmunomodulatory peptide with remarkable anti-inflammatory properties. Daily or twice daily administration of the peptide reduces the symptoms of several inflammatory animal disease models and the peptide has demonstrated safety in human trials. Unfortunately, the pharmacokinetics of peptide delivery are not favorable from the pharmaceutical perspective. For this reason, plasmid-based vectors were created that constitutively express the immunomodulatory peptide. The fusion constructs encode the 13 amino acids of αMSH in frame with the first domain of serum albumin, separated by a linker and furin cleavage sites. The fusion proteins were expressed and processed in human fetal kidney (293) cells. Supernatant from B16/F10 cells transfected with the constructs stimulated secretion of melanin from melanocytes. Furthermore, transfected cytoskeletal muscle (Sol8) cells secreted bioactive αMSH that reduced NF-κB-mediated transcriptional activation of a luciferase reporter gene. The activity of these vectors provides tools and the impetus for testing the constructs in several animal models of chronic inflammation.

Expression and regulation of the meprin ? gene in human cancer cells

A novel mRNA isoform (meprin beta') of the cell-surface protease subunit meprin beta was previously identified in human colon cancer cells. The study reported here revealed that this mRNA isoform was identical within the protein coding region and at the 3' end to the beta isoform of normal intestine but that it contained an extended 5' untranslated region. Meprin beta' mRNA was expressed in the human breast cancer cell lines MCF-7 and SK-BR-3, in the human osteosarcoma cell line U2 Os, and in the human pancreatic cancer cell line BxPC-3. Meprin beta mRNA, but not beta' mRNA, was expressed in human fetal kidney cells. We cloned and sequenced genomic DNA encoding portions of the promoter region of the meprin beta gene. The unique sequences present in the beta' mRNA were present in the human genomic DNA immediately upstream of the transcription start site for the beta mRNA. The human meprin promoter sequence was searched for potential transcription-factor binding sites, and putative activator protein-1, polyoma enhancer activator 3 (PEA3), CCAAT enhancer-binding protein beta, and estrogen-receptor binding sites were identified along with binding sites for the intestine-specific cdx-2 transcription factor. The activity of meprin promoter/luciferase reporter gene constructs transfected into U2 Os cells was highest with constructs containing 83 and 639 bp of promoter DNA. These regions of the promoter each contain a putative PEA3 element. Treatment of the human colon adenocarcinoma cell line HT29-18C1 with 50 or 100 ng/mL phorbol myristal acetate for 8 h increased meprin beta' mRNA levels. Likewise, U2 Os cells transfected with the -639/luciferase or -1800/luciferase constructs showed a phorbol myristal acetate-inducible increase in reporter gene activity, indicating that the PEA3 element within the -639 construct or other elements further upstream respond to phorbol ester.

Expression and regulation of the meprin β gene in human cancer cells

A novel mRNA isoform (meprin β′) of the cell-surface protease subunit meprin β was previously identified in human colon cancer cells. The study reported here revealed that this mRNA isoform was identical within the protein coding region and at the 3′ end to the β isoform of normal intestine but that it contained an extended 5′ untranslated region. Meprin β′ mRNA was expressed in the human breast cancer cell lines MCF-7 and SK-BR-3, in the human osteosarcoma cell line U2 Os, and in the human pancreatic cancer cell line BxPC-3. Meprin β mRNA, but not β′ mRNA, was expressed in human fetal kidney cells. We cloned and sequenced genomic DNA encoding portions of the promoter region of the meprin β gene. The unique sequences present in the β′ mRNA were present in the human genomic DNA immediately upstream of the transcription start site for the β mRNA. The human meprin promoter sequence was searched for potential transcription-factor binding sites, and putative activator protein-1, polyoma enhancer activator 3 (PEA3), CCAAT enhancer-binding protein beta, and estrogen-receptor binding sites were identified along with binding sites for the intestine-specific cdx-2 transcription factor. The activity of meprin promoter/luciferase reporter gene constructs transfected into U2 Os cells was highest with constructs containing 83 and 639 bp of promoter DNA. These regions of the promoter each contain a putative PEA3 element. Treatment of the human colon adenocarcinoma cell line HT29-18C1 with 50 or 100 ng/mL phorbol myristal acetate for 8 h increased meprin β′ mRNA levels. Likewise, U2 Os cells transfected with the -639/luciferase or -1800/luciferase constructs showed a phorbol myristal acetate–inducible increase in reporter gene activity, indicating that the PEA3 element within the -639 construct or other elements further upstream respond to phorbol ester. Mol. Carcinog. 25:169–178, 1999. © 1999 Wiley-Liss, Inc.

Autoantibodies to leukocyte alphaMbeta2 integrin glycoproteins in HIV infection.

HIV infection is often associated with polyclonal B-cell activation, autoantibodies, and clinically evident autoimmune disease. Because neutropenia and anti-neutrophil autoantibodies are common clinical features of HIV disease, we studied a series of HIV+ patients to determine whether anti-alphaMbeta2 integrin (MAC-1) specific anti-neutrophil autoantibodies occur in HIV disease, as we have shown to occur in patients with immune neutropenia not associated with HIV. Two new assays specific for anti-alphaMbeta2 IgG were developed to carry out these studies: an ELISA method using affinity-purified alphaMbeta2 integrin protein, and a flow cytometry method using subclones of the 293 human fetal kidney cell line, stably transfected with cDNAs for the alphaM and/or beta2 integrin subunits. In studies of the sera of 20 untreated HIV+ individuals, anti-alphaMbeta2 activity was detected in 9 (45%) by one or the other of these assays and in 5 (25%) by both assays. Seven of the 20 HIV+ study subjects had unexplained neutropenia, and of these, 6 (86%) were positive for anti-alphaMbeta2 autoantibodies. Our findings indicate that anti-alphaMbeta2 integrin autoantibodies are frequent in HIV+ individuals, particularly when unexplained neutropenia is also present, and raise the possibility that these autoantibodies may have a role in the acquired neutrophil dysfunction and increased risk of nonopportunistic bacterial infections observed in HIV disease.

Placental lactogen-I (PL-I) target tissues identified with an alkaline phosphatase-PL-I fusion protein.

The rat placenta expresses a family of genes related to prolactin (PRL). Target tissues and physiological roles for many members of the PRL family have yet to be determined. In this investigation we evaluated the use of an alkaline phosphatase (AP) tag for monitoring the behavior of a prototypical member of the PRL family, placental lactogen-I (PL-I). A probe was generated consisting of a fusion protein of human placental AP and rat PL-I (AP-PL-I). The AP-PL-I construct was stably expressed in 293 human fetal kidney cells, as was the unmodified AP vector that served as a control. AP activity was monitored with a colorimetric assay in conditioned medium from transfected cells. Immunoreactivity and PRL-like biological activities of the AP-PL-I fusion protein were demonstrated by immunoblotting and the Nb2 lymphoma cell proliferation assay, respectively. AP-PL-I specifically bound to tissue sections known to express the PRL receptor, including the ovary, liver, and choroid plexus. Binding of AP-PL-I to tissues was specific and could be competed with ovine PRL. The results indicate that AP is an effective tag for monitoring the behavior of PL-I and suggest that this labeling system may also be useful for monitoring the actions of other members of the PRL family.

Effects of PAX2 expression in a human fetal kidney (HEK293) cell line

PAX2, a member of the “paired-box” family of homeotic genes, is a nuclear transcription factor expressed in the early stages of nephrogenesis by induced blastemal cells as they progress from mesenchymal condensates to the “S-shaped” stage and also by the ureteric bud. Spontaneous mutations in one copy of PAX2 in humans causes a syndrome of proteinuric renal failure and coloboma of the eye (P. Sanyanusin et al., Nat. Genet. 9 (1995) 358–363); transgenic mice with disruption of the PAX2 gene are anephric (M. Torres et al., Development 121 (1995) 4057–4067. Although PAX2 is clearly critical for normal kidney development, its direct effects on kidney cell phenotype are unknown. To address this issue, we developed stable transfectants of the HEK293 human fetal kidney epithelial cell line expressing human PAX2 protein under tetracycline-regulatable promoter. In these cells, PAX2 had no effect on the proliferative rate, but increased the expression of the Wilms' tumor gene (2-fold) and E-cadherin (7-fold). PAX2 had a strong inhibitory effect on vimentin; vimentin/GAPDH mRNA ratio was suppressed to 8% of control whereas cytokeratin-18/GAPDH mRNA ratio was unchanged. During nephrogenesis, loss of vimentin and onset of low-level WT1 and E-cadherin expression occur in mesenchymal condensates. Our observations suggest that these events may be, in part, regulated by PAX2.

A Far Upstream Cis-element Is Required for Wilms' Tumor-1 (WT1) Gene Expression in Renal Cell Culture

To identify novel cis-regulatory elements responsible for the tissue-restricted expression pattern of the Wilms' tumor-1 (WT1) gene, we mapped a total of 11 DNase I-hypersensitive sites in the 5'-flanking region and first intron of the human gene, six of which were specific for WT1 expressing cell lines. A 1.4-kilobase (kb) fragment from the mouse wt1 5'-flanking region contained cross-hybridizing sequence with significant homology to a region of DNase I hypersensitivity in the human WT1 gene which bound to nuclear matrix in human fetal kidney 293 cells. None of the DNase I-hypersensitive sites/matrix attachment regions, either alone or in combination, were sufficient for tissue-specific WT1 expression in transient and stably transfected cell lines. However, stable transfection of an approximately 620-kb yeast artificial chromosome (YAC) that carried the entire mouse wt1 locus into 293 cells resulted in wt1 (trans)gene expression at a level of approximately 30% of the endogenous human gene. Deletion of the 1.4-kb cross-hybridizing mouse fragment, located approximately 15 kb upstream of the transcription start site, caused complete loss of wt1 gene expression in the YAC-transfected 293 cells. In summary, we have identified a far upstream element that contains a region of DNase I hypersensitivity and that binds to nuclear matrix. This element includes phylogenetically conserved sequence and is required, although not sufficient, for mouse wt1 gene expression in human fetal kidney cells in culture.

Human Butyrylcholinesterase L330I Mutation Belongs to a Fluoride-Resistant Gene, by Expression in Human Fetal Kidney Cells

We noticed a Japanese male showed low serum butyrylcholinesterase(BCHE) activity on health examination. The phenotyping analysis revealed a reduced dibucaine number (DN) and an especially low fluoride number (FN), similar to an FS phenotype. A homozygous missense mutation, a T to A transversion at nucleotide 988, was identified in his BCHE gene. This mutation resulted in the replacement of leucine by isoleucine at codon 330 (L330I). DN and FN of recombinant BCHE(L330I) secreted by human fetal kidney cells were compared to recombinant wild-type(usual gene)BCHE and normal serum BCHE. These results showed this amino acid substitution of BCHE, Leu330 to Ile, really caused the abnormal DN and FN. We conclude that the BCHE L330I mutation is a fluoride-resistant gene, a Japanese type fluoride-resistant gene.

Expression and characterization of isoforms of 3β-hydroxysteroid [formula omitted] in the hamster

The enzyme 3β-hydroxysteroid dehydrogenase Δ 5→4- isomerase (3β-HSD) is essential for the production of all classes of steroid hormones. Multiple isozymes of this enzyme have been demonstrated in the kidney and liver of both the rat and the mouse, although the function of the enzyme in these tissues is unknown. We have characterized three isozymes of 3β-HSD expressed in various tissues of the hamster. Both western and northern blot analyses demonstrated very high levels of 3β-HSD in the adrenal, kidney and male liver. Conversely, there were extremely low levels of enzyme expression in the female liver. cDNA libraries prepared from RNA isolated from hamster adrenal, kidney and liver were screened with a full-length cDNA encoding human type 1 3β-HSD. Separate cDNAs encoding three isoforms of 3β-HSD were isolated from these libraries. To examine the properties of the isoforms, the cDNAs were ligated into expression vectors for over-expression in 293 human fetal kidney cells. The type 1 isoform, isolated from an adrenal cDNA library, was identified as a high-affinity 3β-hydroxysteroid dehydrogenase. A separate isoform, designated type 2, was isolated from the kidney, and this was also a high-affinity dehydrogenase/isomerase. Two cDNAs were isolated from the liver, one identical in sequence to type 2 of the kidney, and a distinct cDNA encoding an isoform designated type 3. The type 3 3β-HSD possessed no steroid dehydrogenase activity but was found to function as a 3-ketosteroid reductase. Thus male hamster liver expresses a high-affinity 3β-HSD (type 2) and a 3-ketosteroid reductase (type 3), whereas the kidney of both sexes express the type 2 3β-HSD isoform. These differ from the type 1 3β-HSD expressed in the adrenal cortex.

Receptor binding of transforming growth factor-β by human fetal adrenal cells

We have shown previously that transforming growth factor β1 (TGF-β1) is antimitotic for human fetal adrenal (HFA) cells in vitro and that this effect can be partially blocked by adrenocorticotropic hormone (ACTH). In the present study, we sought to determine whether ACTH might interfere with TGF-β1 action by means of reducing TGF-β1 binding to adrenal cells. We incubated adrenal cells with 50 pM 125I-labeled TGF-β1 for 15 min to 3 h at 4°C and found that the binding of 125I-labeled TGF-β1 increased with time and could be inhibited in a dose-dependent manner by non-labeled TGF-β1 (0.05–10 nM), but not with other relevant cytokines: IL6, TNFα, IGF-I, IGF-II, TGF-α, and EGR Pretreatment of HFA cells with ACTH (0.009–900 nM) for 4–24 h significantly increased specific 125I-labeled TGF -β1 binding compared to that in untreated cells; maximal increases in binding were achieved with 0.9 nM ACTH. This effect of ACTH could be mimicked by treatment of adrenal cells with dibutyryl CAMP (1 mM) or forskolin (10 μM). Scatchard analysis of data from ACTH-treated cells suggest the presence of two populations of TGF-β1 binding sites with different affinity and capacity of binding for the ligand. The high affinity sites had a K d value of 9.5 × 10 −10 M and a capacity of 5.4 × 10 4 sites/cell, while the low affinity binding structures had a K d value of 4.7 × 10 −8 M and were present in a concentration of 7.6 × 10 5 sites/cell. Up-regulation of TGF-β1 binding by ACTH or its agonists was not observed with cultured human fetal liver or kidney cells. Incubation of HFA cells with cytochalasin D, which causes such cells to round as also occurs with ACTH, had no effect on binding of TGF-β1. Upon cross-linking of the 125I-labeled TGF-β1 bound to HFA cells by sulfosuccinimidyl suberate followed by solubilization and SDS-PAGE electrophoresis, we found TGF-β1 to be associated with three binding sites; the cross-linked complexes had apparent molecular weights of >205, 65 and 45 kDa. Pre-incubation of HFA cells with ACTH increased the binding of labeled TGF -β1 to all three molecular weight substances. In control and ACTH-treated HFA cells (fetal zone and neocortex) the predominant binder was the high molecular weight form. In contrast, the predominant form of TGF-β1 binding in kidney and liver cells had a cross-linked weight of approximately 66 kDa; there was little binding of TGF-β1 to the >205 kDa form. These results indicate that HFA cells have specific binding sites for TGF-β1 that are heterogenous in size. Since ACTH and other agonists of protein kinase A increased adrenal binding of TGF-β1, we suggest that ACTH reversal of TGF-β1 induced inhibition of adrenal growth is apparently not due to decreased binding of TGF-β1.

Mutagenesis of cysteine residues in the human gonadotropin alpha subunit. Roles of individual disulfide bonds in secretion, assembly, and biologic activity.

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common alpha subunit but differ in their hormone-specific beta subunits. Site-directed mutagenesis was used to examine the role of the five disulfide bonds in the alpha subunit on the folding, assembly with the hCG beta subunit, and in cases where dimer formation occurred, receptor binding and signal transduction. Cysteine residues in the disulfide bonds formed by cysteines 7-31, 10-60, 28-82, 59-87, and 32-84 (Lapthorn, A., Harris, D. Littlejohn, A., Lustbader, J. Canfield, R., Machin, K., Morgan, F., and Isaacs, N. (1994) Nature 369, 455-461) were converted to alanine, and these mutants were transfected alone or together with the wild-type hCG beta gene into Chinese hamster ovary cells. The alpha Cys-10, 28, 60, 82, and 84 mutants were not secreted and in most cases were degraded at a faster rate than the native subunit. In addition, these mutants failed to assemble with the hCG beta subunit. Mutants with alterations at alpha Cys-7, 31, 32, 59, or 87 were secreted and combined with the beta subunit. Heterodimers containing a 7-31 double mutant bound to human lutropin-chorionic gonadotropin receptor expressed in transfected human fetal kidney cells, and stimulated cAMP comparable to wild-type hCG. Dimers containing the beta subunit with either single mutant alpha 59, alpha 87, alpha 32, or the alpha 59-87 double mutant showed much lower affinity for the receptor than wild-type hCG. These results suggest that disulfide bonds associated with alpha 7, alpha 31, alpha 59, alpha 87, and alpha 32 are not essential for the alpha subunit to fold into a form that will combine with the hCG beta subunit and to produce a biologically active dimer. This contrasts with observations of the hCG beta subunit where all the disulfide bonds are required for efficient combination and folding (Suganuma, N., Matzuk, M., and Boime, I. (1989) J. Biol. Chem. 264, 19302-19307). In addition, the lack of secretion of some mutants reflects previous observations that proteins which do not fold correctly are rapidly degraded. Thus, alpha subunit mutants which fold properly are secreted and can form heterodimers.