Secretoglobin 2A1 is under selective androgen control mediated by a peculiar binding site for Sp family transcription factors

Human secretoglobin (SCGB) 2A1 (or lipophilin C, lacryglobin, mammaglobin B) is a small protein of unknown function that forms heterodimers with secretoglobin 1D1 (lipophilin A) in tears and is expressed in the prostate. Here we show that SCGB 2A1 is under androgen control in the androgen-responsive prostatic cell line LNCaP and can be induced more than 20-fold by dihydrotestosterone. Only 6 h after androgen treatment, a strong DNase I-hypersensitive site is induced in the proximal promoter within chromatin. Within the boundaries of this DNase I-hypersensitive site a minimal 32-bp peculiar dimeric inverted repeat variant GC box (dim-IR-GA box) was found to confer androgen but not glucocorticoid responsiveness in gene transfer experiments. Mutations of both GA boxes that abolish binding of Sp1 and Sp3 also abrogate the androgen response. In an EMSA the DNA binding domain of the androgen receptor (AR) was not able to bind directly to the dim-IR-GA box. However, AR is functionally required for the hormone response because induction can be inhibited with the nonsteroidal antagonist bicalutamide. Chromatin immunoprecipitation experiments demonstrated that AR is recruited to the proximal promoter 10 min after androgen treatment. Therefore we propose that SCGB 2A1 represents a new class of androgen target genes that are purely under indirect AR control mediated by DNA-bound Sp factors.     

Lipophilins: human peptides homologous to rat prostatein

Lipophilin components A, B and C are human homologues of prostatein, the major secreted protein of rat prostate. This report describes their cDNA sequences, tissue expression and chromosomal localization. Lipophilin gene products were widely expressed in normal tissues, especially in endocrine-responsive organs. The gene for lipophilin C (also called mammaglobin b) is located on chromosome 11q12-q13.1, near the mammaglobin gene, a homologue overexpressed in many breast cancers. The lipophilin B gene resides on chromosome 10q23, a region deleted in many tumors, and the lipophilin A gene is on chromosome 15q12-q13.     

Mammaglobin is found in breast tissue as a complex with BU101

The mammaglobin gene has been shown to be preferentially expressed in breast tissue. Few genes match its specificity. Mammaglobin has generated much interest, and studies are ongoing to develop diagnostic tests for breast cancer based on the detection of mammaglobin. While searching the Incyte Genomics Lifeseq database for tissue-specific markers, we observed a second secretoglobin, BU101, also known as lipophilin B. We report here that mammaglobin, in breast tissue, is found as a complex with BU101. The complex was isolated from breast cancer tissue and was characterized as the biologically relevant form of mammaglobin.     

Purification and characterization of the mammaglobin/lipophilin B complex,a promising diagnostic marker for breast cancer

Mammaglobin, a promising diagnostic marker for breast cancer, forms a covalent complex with lipophilin B. mRNA levels for each component of the complex were determined for a number of breast tumors and normal tissues, and correlation of message expression was highly significant between mammaglobin and lipophilin B (p < 0.0001). The complex was purified by both standard biochemical techniques and immunoaffinity chromatography. N-Terminal sequencing revealed that mammaglobin and lipophilin B are processed as predicted by cleavage of their signal sequence after amino acids 19 and 21, respectively. Three molecular masses-representing the fully glycosylated form, the complex without one of the carbohydrate chains, and the deglycosylated proteins-are detected by ProteinChip array SELDI-TOF mass spectrometry after partial enzymatic deglycosylation. This is consistent with the two predicted N-linked glycosylation sites in the primary sequence of mammaglobin and each site having an attached sugar of approximately 3500 Da. Reducing agents release lipophilin B from mammaglobin, and the free peptides are seen at their predicted molecular masses in the deglycosylated complex. Molecular modeling, secondary structure prediction, and circular dichroism indicate that the complex is a small alpha-helical globule that has three disulfide bridges and a carbohydrate chain at each pole. LC-ESI-MS shows that mammaglobin and lipophilin B are bonded in a head to tail orientation. This work describes the biochemistry of the mammaglobin/lipophilin B complex and lays the framework for use of this complex as a novel protein-based serological marker for breast cancer.     

Secretoglobin 3A2 suppresses bleomycin-induced pulmonary fibrosis by transforming growth factor beta signaling down-regulation

With increasing worldwide rates of morbidity and mortality of pulmonary fibrosis, the development of effective therapeutics for this disease is of great interest. Secretoglobin (SCGB) 3A2, a novel cytokine-like molecule predominantly expressed in pulmonary airways epithelium, exhibits anti-inflammatory and growth factor activities. In the current study SCGB3A2 was found to inhibit TGFβ-induced differentiation of fibroblasts to myofibroblasts, a hallmark of the fibrogenic process, using pulmonary fibroblasts isolated from adult mice. This induction was through increased phosphorylation of STAT1 and expression of SMAD7 and decreased phosphorylation of SMAD2 and SMAD3. To demonstrate the effect of SCGB3A2 on the TGFβ signaling in vivo, a bleomycin-induced pulmonary fibrosis mouse model was used. Mice were administered bleomycin intratracheally followed by intravenous injection of recombinant SCGB3A2. Histological examination in conjunction with inflammatory cell counts in bronchoalveolar lavage fluids demonstrated that SCGB3A2 suppressed bleomycin-induced pulmonary fibrosis. Microarray analysis was carried out using RNAs from lungs of bleomycin-treated mice with or without SCGB3A2 and normal mice treated with SCGB3A2. The results demonstrated that SCGB3A2 affects TGFβ signaling and reduces the expression of genes involved in fibrosis. This study suggests the potential utility of SCGB3A2 for targeting TGFβ signaling in the treatment of pulmonary fibrosis.     

Farnesyl diphosphate synthase is abundantly expressed and regulated by androgen in rat prostatic epithelial cells

Farnesyl diphosphate synthase (FPPS) has been identified as an androgen-response gene in the rat ventral prostate using a highly sensitive PCR-based cDNA subtraction technique. FPPS is an essential enzyme that catalyzes the synthesis of farnesyl diphosphate (FPP), which is required for cholesterol biosynthesis as well as protein prenylation. We have characterized the expression of FPPS in the rat prostate in response to androgen manipulation. Northern blot analysis showed that castration induced a 10-fold down-regulation of FPPS mRNA within 24 h in the ventral prostate and androgen replacement up-regulated FPPS mRNA rapidly in the regressed ventral prostate of a castrated rat. The expression of FPPS was also regulated by androgen in the lateral and dorsal prostate, indicating that FPPS is important to androgen action in all three lobes of the prostate. Western blot analysis showed that FPPS protein level was also regulated by androgen in the prostate. Northern blot analysis of tissue specificity indicated that FPPS was most abundantly expressed in the ventral prostate of a mature rat and was responsive to androgen manipulation in the prostate and seminal vesicles, but not in other tissues. In situ hybridization study showed that FPPS mRNA was localized to the prostatic epithelium. Interestingly, the expression of FPPS was elevated in Dunning rat prostate tumor cell lines. The above findings suggest that FPPS has the potential to play an important role in androgen action and prostate cancer progression.     

Mammaglobin in peripheral blood and the tumor of breast cancer patients

Currently, no molecular biological markers do exist for early diagnosis of breast cancer. One of the possible candidates for the marker of early breast cancer is mammaglobin (MGB1) or SCGB2A2 (secretoglobin, family 2A, member 2), characterized by the maximal expression level in early breast cancer. Using the RT-PCR method MGB1 mRNA expression was examined in 57 tumor tissue samples and 57 samples of morphologically non-malignant tissue (MNT) of breast cancer (BC) patients. Specificity and sensitivity of the MGB1 mRNA assay in peripheral blood of BC patients was evaluated by nested PCR. 169 blood samples (from 95 BC patients, 22 from patients with benign breast tumors, 28 from patients with tumors of other localizations, and 24 samples from healthy donors) have been analyzed. MGB1 expression was significantly higher in BC tissue samples compared to MNT (p = 0.0019). The maximal expression level was in the samples T1 (p = 0.013), stage I BC (p = 0.037), GI (p = 0.0019). MGB1 expression positively correlated with expression of estrogen (p = 0.034) and progesterone (p = 0.0004) receptors. Sensitivity and specificity of the MGB1 mRNA assay in peripheral blood were 60.6 and 92.3%, respectively. Expression of MGB1 was higher in BC than MNT and it decreased during BC progression. The sensitivity and specificity of the MGB1 mRNA assay may be used as an additional diagnostic method.     

Cloned proteolipid protein and myelin basic protein cDNA. Transcription of the two genes during myelination

cDNA clones of rat brain proteolipid protein (PLP), also named lipophilin, the major integral myelin membrane protein, and of myelin basic protein (MBP), the major extrinsic myelin protein, have been isolated from a rat brain cDNA library cloned into the PstI site of pBR322. Poly(A)+ RNA from actively myelinating 18-day-old rats has been reversely transcribed. Oligonucleotides synthesized according to the established amino-acid sequence of lipophilin and the nucleotide sequence of the small myelin basic protein of the N-terminal, the central and C-terminal region of their sequences were used as hybridization probes for screening. The largest insert in one of several lipophilin clones was 2,585 base pairs (bp) in length (pLp 1). It contained 521 bp of the C-terminal coding sequence and the complete 2,064 bp long non-coding 3' sequence. The myelin basic protein cDNA insert of clones pMBP5 and pMBP6 is 2,530 bp long and that of clones pMBP2 and pMBP3 640 bp. These clones were also characterized. pMBP2 was sequenced and used together with the lipophilin cDNA clones as hybridization probes to estimate the lipophilin and myelin basic protein mRNA levels of rat brain during the myelination period. The expression of the lipophilin and myelin basic protein genes during development of the myelin sheath appears to be strictly coordinated.     

Dual role of FoxA1 in androgen receptor binding to chromatin, androgen signalling and prostate cancer

High androgen receptor (AR) level in primary tumour predicts increased prostate cancer-specific mortality. However, the mechanisms that regulate AR function in prostate cancer are poorly known. We report here a new paradigm for the forkhead protein FoxA1 action in androgen signalling. Besides pioneering the AR pathway, FoxA1 depletion elicited extensive redistribution of AR-binding sites (ARBs) on LNCaP-1F5 cell chromatin that was commensurate with changes in androgen-dependent gene expression signature. We identified three distinct classes of ARBs and androgen-responsive genes: (i) independent of FoxA1, (ii) pioneered by FoxA1 and (iii) masked by FoxA1 and functional upon FoxA1 depletion. FoxA1 depletion also reprogrammed AR binding in VCaP cells, and glucocorticoid receptor binding and glucocorticoid-dependent signalling in LNCaP-1F5 cells. Importantly, FoxA1 protein level in primary prostate tumour had significant association to disease outcome; high FoxA1 level was associated with poor prognosis, whereas low FoxA1 level, even in the presence of high AR expression, predicted good prognosis. The role of FoxA1 in androgen signalling and prostate cancer is distinctly different from that in oestrogen signalling and breast cancer.