HSPA2 Protein Research Articles

Evaluation of seminal plasma HSPA2 protein as a biomarker of human spermatogenesis status

In mammals, testicular Heat shock-related 70 kDa protein 2 (HSPA2) is a chaperon strictly linked to spermatogenesis status, whereas its presence in spermatozoa ensures successful oocyte fertilization. However, there is little information on this protein in seminal plasma in infertile males. Based on our previous two independent studies, we have selected HSPA2 to evaluate this seminal plasma protein is a potential biomarker of correct spermatogenesis.Using immunoblotting and mass spectrometry (MS) we have screened human seminal plasma samples for the presence of HSPA2. Samples were obtained from individuals with normozoospermia, cryptozoospermia, non-obstructive and obstructive azoospermia.Our results showed a lack of HSPA2 in seminal plasma in all azoospermic males however, in cryptozoospermia the results were extremely diversified. Additionally, the application of 2-dimensional gel electrophoresis (2-DE) indicated the presence of additional protein isoforms suggesting possible mechanisms underlying the male infertility.Our findings suggest seminal plasma HSPA2 protein as a possible biomarker not only of spermatogenesis status, especially in cryptozoospermic males, but also as a biomarker predicting the success of reproductive treatment including assisted reproductive techniques (ART).

Transcriptomic analysis identifies a tumor subtype mRNA classifier for invasive non-functioning pituitary neuroendocrine tumor diagnostics.

Rationale: The invasive behavior of non-functioning pituitary neuroendocrine tumors (NF-PitNEts) presents obstacles for complete surgical resection and is indicative of poor prognosis. Therefore, developing reliable diagnostic tools for identifying invasive PitNEts would be helpful in guiding surgical decisions and, in particular, the follow-up treatment.Methods: We analyzed differential gene expression profiles between 39 non-invasive and 22 invasive NF-PitNEts by high-throughput sequencing, gene co-expression, and functional annotation. Twenty-one transcripts were further validated by Taqman-qPCR in another 143 NF-PitNEt samples. The histological expression and serum-exosomal mRNA of three candidate genes were examined by tissue microarray and droplet digital PCR.Results: Non-invasive and invasive NF-PitNEts were clustered into distinct groups with a few outliers because of their gonadotroph, corticotroph, or null cell lineages. The gene signature with strong invasive potential was enriched in 'Pathways in cancers' and 'MAPK pathway', with significantly higher in situ INSM1 and HSPA2 protein expression in invasive NF-PitNEts. Further integration of the 20 qPCR-validated differentially expressed genes and pituitary cell lineages provided a gene-subtype panel that performed 80.00-90.24% diagnostic accuracy for the invasiveness of NF-PitNEts.Conclusion: Our approach defined new characteristics in the core molecular network for patients at risk for invasive NF-PitNEt, representing a significant clinical advance in invasive PitNEt diagnostics.

Genetic background dependent modifiers of craniosynostosis severity

Craniosynostosis severity varies in patients with identical genetic mutations. To understand causes of this phenotypic variation, we backcrossed the FGFR2+/C342Y mouse model of Crouzon syndrome onto congenic C57BL/6 and BALB/c backgrounds. Coronal suture fusion was observed in C57BL/6 (88% incidence, p < .001 between genotypes) but not in BALB/c FGFR2+/C342Y mutant mice at 3 weeks after birth, establishing that that the two models differ in phenotype severity. To begin identifying pre-existing modifiers of craniosynostosis severity, we compared transcriptome signatures of cranial tissues from C57BL/6 vs. BALB/c FGFR2+/+ mice. We separately analyzed frontal bone with coronal suture tissue from parietal bone with sagittal suture tissues because the coronal suture but not the sagittal suture fuses in FGFR2+/C342Y mice. The craniosynostosis associated Twist and En1 transcription factors were down-regulated, while Runx2 was up-regulated, in C57BL/6 compared to BALB/c tissues, which could predispose to craniosynostosis. Transcriptome analyses under the GO term MAPK cascade revealed that genes associated with calcium ion channels, angiogenesis, protein quality control and cell stress response were central to transcriptome differences associated with genetic background. FGFR2 and HSPA2 protein levels plus ERK1/2 activity were higher in cells isolated from C57BL/6 than BALB/c cranial tissues. Notably, the HSPA2 protein chaperone is central to craniofacial genetic epistasis, and we find that FGFR2 protein is abnormally processed in primary cells from FGFR2+/C342Y but not FGFR2+/+ mice. Therefore, we propose that differences in protein quality control responses may contribute to genetic background influences on craniosynostosis phenotype severity.

Functional redundancy of HSPA1, HSPA2 and other HSPA proteins in non-small cell lung carcinoma (NSCLC); an implication for NSCLC treatment

Heat shock proteins (HSPs) are a large group of chaperones considered critical for maintaining cellular proteostasis. Their aberrant expression in tumors can modulate the course of processes defined as hallmarks of cancer. Previously, we showed that both stress-inducible HSPA1 and testis-enriched HSPA2, highly homologous members of the HSPA (HSP70) family, are often overexpressed in non-small cell lung carcinoma (NSCLC). HSPA1 is among the best characterized cancer-related chaperones, while the significance of HSPA2 for cancer remains poorly understood. Previously we found that in primary NSCLC, HSPA1 was associated with good prognosis while HSPA2 correlated with bad prognosis, suggesting possible different roles of these proteins in cancer. Therefore, in this work we investigated the impact of HSPA1 and HSPA2 on NSCLC cell phenotype. We found that neither paralog-selective nor simultaneous knockdown of HSPA1 and HSPA2 gene expression reduced growth and chemoresistance of NSCLC cells. Only blocking of HSPA proteins using pan-HSPA inhibitors, VER-155008 or JG-98, exerted potent anticancer effect on NSCLC cells, albeit the final outcome was cell type-dependent. Pan-HSPA inhibition sensitized NSCLC cells to bortezomib, but not to platinum derivates. Our result suggests the inhibitors of proteasome and HSPAs seem an effective drug combination for pre-clinical development in highly aggressive NSCLC.

Novel role for the testis-enriched HSPA2 protein in regulating epidermal keratinocyte differentiation.

HSPA2, a poorly characterized member of the HSPA (HSP70) chaperone family, is a testis-enriched protein involved in male germ cell differentiation. Previously, we revealed that HSPA2 is present in human stratified epithelia, including epidermis, however the contribution of this protein to epithelial biology remained unknown. Here, we show for the first time that HSPA2 is expressed in basal epidermal keratinocytes, albeit not in keratinocytes exhibiting features attributed to primitive undifferentiated progenitors, and participates in the keratinocyte differentiation process. We found that HSPA2 is dispensable for protection of HaCaT keratinocytes against heat shock-induced cytotoxicity. We also shown that lentiviral-mediated shRNA silencing of HSPA2 expression in HaCaT cells caused a set of phenotypic changes characteristic for keratinocytes committed to terminal differentiation such as reduced clonogenic potential, impaired adhesiveness and increased basal and confluency-induced expression of differentiation markers. Moreover, the fraction of undifferentiated cells that rapidly adhered to collagen IV was less numerous in HSPA2-deficient cells than in the control. In a 3D reconstructed human epidermis model, HSPA2 deficiency resulted in accelerated development of a filaggrin-positive layer. Collectively, our results clearly show a link between HSPA2 expression and maintenance of keratinocytes in an undifferentiated state in the basal layer of the epidermis. It seems that HSPA2 could retain keratinocytes from premature entry into the terminal differentiation process. Overall, HSPA2 appears to be necessary for controlling development of properly stratified epidermis and thus for maintenance of skin homeostasis.

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmdmdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca2+ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.

Reporter mice express green fluorescent protein at initiation of meiosis in spermatocytes

Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co-localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post-meiotic germ cells and not in others. Fluorescence activated cell sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2-GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post-meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage-specific spermatogenic cell proteins and developmental events.

Expression, function, and regulation of the testis-enriched heat shock HSPA2 gene in rodents and humans.

The HSPA2 gene is a poorly characterized member of the HSPA (HSP70) family. HSPA2 was originally described as testis-specific and expressed at the highest level in pachytene spermatocytes of rodents, the expression of which is not induced by heat shock. HSPA2 is crucial for male fertility. However, recent advances have shown that HSPA2 is expressed in various tumors and in certain types of somatic tissues. In this review, we summarize the current knowledge on the HSPA2 expression pattern, including information on transcriptional, translational, posttranslational, and epigenetic mechanisms which regulate HSPA2 expression. We also present and discuss the current views concerning the functions of the HSPA2 protein in spermatogenetic, somatic, and cancer cells. The knowledge of the properties of HSPA2, although limited, shows this protein as a unique member of the HSPA family. However, understanding whether this protein could become a relevant cancer biomarker or a therapeutically applicable target requires extensive further studies.

Interfacial Behavior of Murine Pulmonary Surfactant Expressing Different Human Surfactant Protein SP-A Variants

Pulmonary surfactant is a lipoprotein complex which reduces surface tension to prevent alveolar collapse. At the same time, surfactant also contributes to the protection of the large respiratory surface from the entry of pathogens. It is composed of around 92% lipids and 8% of specific surfactant proteins by mass. One of these proteins is SP-A, a hydrophilic glycoprotein of the collectin family. The main function of this protein is related with host defense. However, previous studies showed that SP-A also aids in the formation and surface behavior of surfactant films at the air-water interface. Humans possess two SP-A genes expressing protein variants SP-A1 and SP-A2. Although their specific or combined activity is not well understood, it has been shown that both gene products are necessary for tubular myelin formation, an extracellular structural form of surfactant. The goal of the present study was to investigate potential differences in the biophysical behavior of surfactants containing human SP-A1, SP-A2, or both. For this purpose we have studied pulmonary surfactant from humanized transgenic mice expressing hSP-A1, hSP-A2, or both human SP-As instead of the endogenous murine protein, in the Captive Bubble Surfactometer (CBS), which simulates alveolar interfacial compression-expansion dynamics. We observed that surfactant containing both hSP-A1 and hSP-A2 reaches lower surface tension after initial and post-expansion interfacial adsorption than surfactants containing only hSP-A1 or hSP-A2. Under interfacial cycling conditions (Quasi-static or Dynamic compression-expansion regimes) surfactant films containing both hSP-A1 and hSP-A2 also showed better performance than surfactant containing only SP-A1 or SP-A2. We conclude that the simultaneous presence of hSP-A1 and hSP-A2 proteins permits surfactant to adopt a particularly favorable structure with optimal functional properties.NIH HL34788

HSPA2 overexpression protects V79 fibroblasts against bortezomib-induced apoptosis

Human HSPA2 is a member of the HSPA (HSP70) family of heat-shock proteins, encoded by the gene originally described as testis-specific. Recently, it has been reported that HSPA2 can be also expressed in human somatic tissues in a cell-type specific manner. The aim of the present study was to find out whether HSPA2 can increase the resistance of somatic cells to the toxic effect of heat shock, proteasome inhibitors, and several anticancer cytostatics. We used a Chinese hamster fibroblast V79 cell line because these cells do not express the HSPA2 and cytoprotective HSPA1 proteins under normal culture conditions and show limited ability to express HSPA1 in response to heat shock and proteasome inhibitors. We established, by retroviral gene transfer, a stable V79/HSPA2 cell line, which constitutively overexpressed HSPA2 protein. The major observation of our study was that HSPA2 increased long-term survival of cells subjected to heat shock and proteasome inhibitors. We found, that HSPA2 confers resistance to bortezomib-induced apoptosis. Thus, we showed for the first time that in somatic cells HSPA2 can be a part of a system protecting cells against cytotoxic stimuli inducing proteotoxic stress.

Differential expression of HSPA1 and HSPA2 proteins in human tissues; tissue microarray-based immunohistochemical study

In the present study we determined the expression pattern of HSPA1 and HSPA2 proteins in various normal human tissues by tissue-microarray based immunohistochemical analysis. Both proteins belong to the HSPA (HSP70) family of heat shock proteins. The HSPA2 is encoded by the gene originally defined as testis-specific, while HSPA1 is encoded by the stress-inducible genes (HSPA1A and HSPA1B). Our study revealed that both proteins are expressed only in some tissues from the 24 ones examined. HSPA2 was detected in adrenal gland, bronchus, cerebellum, cerebrum, colon, esophagus, kidney, skin, small intestine, stomach and testis, but not in adipose tissue, bladder, breast, cardiac muscle, diaphragm, liver, lung, lymph node, pancreas, prostate, skeletal muscle, spleen, thyroid. Expression of HSPA1 was detected in adrenal gland, bladder, breast, bronchus, cardiac muscle, esophagus, kidney, prostate, skin, but not in other tissues examined. Moreover, HSPA2 and HSPA1 proteins were found to be expressed in a cell-type-specific manner. The most pronounced cell-type expression pattern was found for HSPA2 protein. In the case of stratified squamous epithelia of the skin and esophagus, as well as in ciliated pseudostratified columnar epithelium lining respiratory tract, the HSPA2 positive cells were located in the basal layer. In the colon, small intestine and bronchus epithelia HSPA2 was detected in goblet cells. In adrenal gland cortex HSPA2 expression was limited to cells of zona reticularis. The presented results clearly show that certain human tissues constitutively express varying levels of HSPA1 and HSPA2 proteins in a highly differentiated way. Thus, our study can help designing experimental models suitable for cell- and tissue-type-specific functional differences between HSPA2 and HSPA1 proteins in human tissues.Electronic supplementary materialThe online version of this article (doi:10.1007/s00418-011-0791-5) contains supplementary material, which is available to authorized users.

The HspA2 protein localizes in nucleoli and centrosomes of heat shocked cancer cells

The human HSPA2 gene, which belongs to the HSP70 family of heat shock genes, is a counterpart of rodent testis-specific HspA2 gene. Rodent genes are expressed mainly in pachytene spermatocytes, while transcripts of human HSPA2 gene have been detected in various normal somatic tissues, albeit translation of the messenger RNA into corresponding protein has not been yet unambiguously demonstrated, except for several cancer cell lines. The aim of our work, a first step in search for HspA2 function in cancer cells, was to establish its intracellular localization at physiological temperature and during heat shock. First, we used qRT-PCR and a highly specific antibody to select cell lines with the highest expression of the HspA2 protein, which turned out to be A549 and NCI-H1299 lines originating from non-small cell lung carcinoma (NSCLC). Significant expression of the HspA2 was also detected by immunohistochemistry in primary NSCLC specimens. Intracellular localization of the HspA2 was studied using both the specific anti-HspA2 polyclonal antibody and transfection of cells with fusion proteins HspA2-EGFP and mRFP-HspA2. We found that, at physiological temperature, the HspA2 was localized primarily in cytoplasm whereas, during heat shock, localization shifted to nucleus and nucleoli. Moreover, we demonstrate that in heat-shocked cells HspA2 accumulated in centrosomes. Our results suggest that the HspA2, like Hsp70 protein, can be involved in protecting nucleoli and centrosomes integrity in cancer cells subjected to heat shock and, possibly, other cellular stressors.

Identification of heat shock protein 70 genes HSPA2, HSPA5 and HSPA8 from the Japanese quail, Coturnix japonica

ABSTRACTWe have identified three members of the heat shock protein (HSP) 70 family from the Japanese quail, Coturnix japonica. The coding regions of the genes, CjHSPA2, CjHSPA5 and CjHSPA8, exhibit more than 96% similarity to related genes in the chicken, GgHSP70, GgHSPA5 and GgHSC70. The putative proteins encoded by the quail genes contain motifs considered essential for HSP70 family members. In addition, the predicted proteins are more similar to HSPA2, HSPA5, and HSPA8 proteins from vertebrates than they are to other members of the HSP70 family. As is true for orthologs from chicken and mammals, the quail genomic loci consist of a single exon (CjHSPA2) or eight exons (CjHSPA5 and CjHSPA8). The promoter regions of the genes also contain conserved motifs and are similar in structure to promoter regions of related genes in other species. Furthermore, gene order in the regions surrounding the quail HSP70 has been preserved, providing evidence that the genomic regions are orthologous to HSP70‐containing regions in the chicken genome. One of the quail HSP70‐related genes, CjHSPA2, exhibited a robust response to heat shock treatment and transcription of CjHSPA8 mRNA was weakly induced by heat treatment. Taken together, the data suggest that CjHSPA2, CjHSPA5 and CjHSPA8 encode orthologs of HSPA2, HSPA5 and HSPA8 with the notable difference that CjHSPA2 and CjHSPA8 are heat shock‐inducible genes, whereas HSPA2 and HSPA8 are constitutively expressed in a heat shock‐independent manner.

Specific expression of heat shock protein HspA2 in human male germ cells.

In the mouse, the heat shock protein 70-2 (Hsp70-2) has been found to play a critical role in spermatogenesis. The HspA2 gene is the human homologue of the murine Hsp70-2 gene with 91.7% identity in the nucleotide coding sequence. We examined the expression of HspA2 in human tissues. To detect HspA2 expression, antiserum 2A that was raised against mouse Hsp70-2 and that cross-reacted with human HspA2 protein expressed in Escherichia coli was used. The results of Western blotting indicate that significant HspA2 expression occurs in testes with normal spermatogenesis, whereas only a low amount of HspA2 was expressed in testis with Sertoli cell-only syndrome. Only a small amount of HspA2 was detected in breast, stomach, prostate, colon, liver, ovary, and epididymis. Immunoreactivity to HspA2 was present in spermatocytes and spermatids in the testes with normal spermatogenesis, while immunoreactivity to HspA2 in testis with Sertoli cell-only syndrome was remarkably decreased or inconspicuous over the entire cell. These results demonstrate that the HspA2 protein is highly expressed in human male specific germ cells, suggesting that HspA2 protein may play a specific role during meiosis in human testes as found in the murine model.