Sex-specific response of rat costochondral cartilage growth plate chondrocytes to 17ß-estradiol involves differential regulation of plasma membrane associated estrogen receptors.

Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17ß-estradiol (E2) induces membrane responses leading to activation of phospholipase A2 (PLA2), phospholipase C (PLC), prostaglandin E2 (PGE2) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. This study investigated if these sex-specific responses are due to differences in the actual ERs or in downstream signaling. Western blots and flow cytometry of costochondral cartilage resting zone chondrocytes (RCs) showed 2-3 times more ERα in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E2-dependent ER-translocation to the PM, indicating palmitoylation was required. Co-immunoprecipitation showed E2 induced complex formation between ER isoforms only in female RCs. To examine if the lack of response in PKC and PGE2 in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA2. Selective ERα (propylpyrazole triol, PPT) and ERß (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E2's effect on PKC and the cytosolic PLA2 inhibitor, AACOCF3 inhibited the effect on PGE2 in female RCs, confirming involvement of PLC and PLA2 in the mechanism. The PLC activator, m-3M3FßS activated PKC and PLAA peptide increased PGE2 levels in male and female RCs, showing that the signaling pathways are present. These data indicate that differences in membrane ER amount, localization, translocation and interaction are responsible for the sexual dimorphic response to E2.

Sex-specific effects of 17ß-estradiol and dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERß and require palmitoylation to translocate the receptors to the plasma membrane.

Rat costochondral cartilage growth plate chondrocytes exhibit cell sex-specific responses to 17ß-estradiol (E2), testosterone, and dihydrotestosterone (DHT). Mechanistically, E2 and DHT stimulate proliferation and extracellular matrix synthesis in chondrocytes from female and male rats, respectively, by signaling through protein kinase C (PKC) and phospholipase C (PLC). Estrogen receptors (ERα; ERß) and androgen receptors (ARs) are present in both male and female cells, but it is not known whether they interact to elicit sex-specific signaling. We used specific agonists and antagonists of these receptors to examine the relative contributions of ERs and ARs in membrane-mediated E2 signaling in female chondrocytes and DHT signaling in male chondrocytes. PKC activity in female chondrocytes was stimulated by agonists of ERα and ERß and required intact caveolae; PKC activity was inhibited by the E2 enantiomer and by an inhibitor of ERß. Western blots of cell lysates co-immunoprecipitated for ERα suggested the formation of a complex containing both ERα and ERß with E2 treatment. DHT and DHT agonists activated PKC in male cells, while AR inhibition blocked the stimulatory effect of DHT on PKC. Inhibition of ERα and ERß also blocked PKC activation by DHT. Western blots of whole-cell lysates, plasma membranes, and caveolae indicated the translocation of AR to the plasma membrane and specifically to caveolae with DHT treatment. These results suggest that E2 and DHT promote chondrocyte differentiation via the ability of ARs and ERs to form a complex. The results also indicate that intact caveolae and palmitoylation of the membrane receptor(s) or membrane receptor complex containing ERα and ERß is required for E2 and DHT membrane-associated PKC activity in costochondral cartilage cells.

Recreating composition, structure, functionalities of tissues at nanoscale for regenerative medicine.

Nanotechnology offers significant potential in regenerative medicine, specifically with the ability to mimic tissue architecture at the nanoscale. In this perspective, we highlight key achievements in the nanotechnology field for successfully mimicking the composition and structure of different tissues, and the development of bio-inspired nanotechnologies and functional nanomaterials to improve tissue regeneration. Numerous nanomaterials fabricated by electrospinning, nanolithography and self-assembly have been successfully applied to regenerate bone, cartilage, muscle, blood vessel, heart and bladder tissue. We also discuss nanotechnology-based regenerative medicine products in the clinic for tissue engineering applications, although so far most of them are focused on bone implants and fillers. We believe that recent advances in nanotechnologies will enable new applications for tissue regeneration in the near future.

Role of ERα36 in membrane-associated signaling by estrogen.

Traditionally, steroid hormones such as the vitamin D3 metabolites, testosterone and dihydrotesterone, and 17ß-estradiol act through cytosolic and nuclear receptors that directly interact with DNA to alter gene transcription and regulate cellular development. However, recent studies focused on rapid and membrane effects of steroid hormones have given invaluable insight into their non-classical mechanisms of action. In some cases, the traditional receptors were implicated as acting also in the plasma membrane as membrane-associated receptors. However, recent data have demonstrated the presence of an alternative splicing variant to traditional estrogen receptor α known as ERα36, which is present in the plasma membranes of several different cell types including several cancer cell types and even in some normal cells including cartilage and bone cells. The physiological effects that result from the membrane activation of ERα36 may vary from one cell type to another, but the mechanism of action appears to use similar pathways such as the activation of various protein kinases and phospholipases leading to the activation of signaling cascades that result in rapid, non-genomic responses. These rapid responses can affect cell proliferation and apoptotic signaling, indirectly activate downstream genomic signaling through phosphorylation cascades of transcription factors, and crosstalk with classical pathways via interaction with classical receptors. This review describes the data from the last several years and discusses the non-classical, rapid, and membrane-associated cellular responses to steroid hormones, particularly 17ß-estradiol, through the classical receptors ERα and ERß and various non-classical receptors, especially estrogen receptor-α36 (ERα36).

Rapid membrane responses to dihydrotestosterone are sex dependent in growth plate chondrocytes.

Sex steroids are important regulators for longitudinal growth, bone mass accrual, and sexual dimorphism of the skeleton. 17ß-Estradiol regulates proliferation and differentiation of female chondrocytes via a membrane-associated signaling pathway in addition to its estrogen receptor (ER) mediated effects. In contrast, testosterone does not elicit a similar membrane response, either in male or female cells. Whereas female rat growth plate chondrocytes convert testosterone to 17ß-estradiol, male chondrocytes produce 5α-dihydrotestosterone (DHT). Previously DHT was found to mediate sex-specific effects of testosterone in male cells, but it is not known if a membrane-signaling pathway is involved. In this study, we hypothesized that DHT can induce sex-specific rapid membrane effects similar to other steroid hormones. Confluent cultures of chondrocytes isolated from resting zones of growth plates of both male and female rats were treated with 10(-10)-10(-7)M testosterone or DHT for 3, 9, 90 and 270min and protein kinase C (PKC) and phospholipase A2 (PLA2) activities were measured. To examine potential signaling pathways involved in PKC activation, male chondrocytes were treated with 10(-7)M DHT for 9min in the presence or absence of the phospholipase C (PLC) inhibitor U73122, the secretory PLA2 inhibitor quinacrine or the cytosolic PLA2 inhibitor AACOCF3; the Gαi inhibitor pertussis toxin (PTX) or Gαs activator cholera toxin (CTX), and the general G-protein inhibitor GDPßS; thapsigargin, an inhibitor of a Ca-ATPase pump in the endoplasmic reticulum; verapamil and nifedipine, inhibitors of specific L type Ca2+ channels on the cell membrane; and cyproterone acetate (CPA), which is an inhibitor of the classical androgen receptor (AR); as well as the transcription inhibitor actinomycin D, or the translation inhibitor cycloheximide. DHT induced a dose-dependent increase in PKC and PLA2 activity in male cells with the highest increase at 10(-7)M DHT (p<0.05), whereas testosterone had no effect. PKC activity was augmented at 9 and 90 min, and then decreased to baseline at 270min. Neither testosterone nor DHT affected PKC in female cells. U73122, quinacrine, and AACOCF3 inhibited DHT-induced activation of PKC. DHT treatment for 9 min had no effect in [(3)H]-thymidine incorporation in quiescent confluent cultures but caused a dose dependent increase in alkaline phosphatase specific activity. Inhibition of PLC reduced the response of to DHT in a dose dependent manner, indicating that PLC is involved. In conclusion, our study indicates that DHT, but not testosterone, has sex-specific rapid membrane effects in male growth plate chondrocytes involving PLC and PLA2-mediated PKC signaling pathways. Together with previous observations showing that male cells convert testosterone to DHT, these results suggest that DHT might act in the membrane through an autocrine/paracrine mechanism.